Isolated nucleic acid molecules encoding Smad7

ABSTRACT

The invention describes nucleic acids encoding the Smad7 protein, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such nucleic acids, and antibodies relating thereto. Methods and products for using such nucleic acids and polypeptides also are provided.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 from U.S.provisional application serial No. 60/047,221, filed May 20, 1997, fromU.S. provisional application serial No. 60/060,465, filed Sep. 30, 1997,from U.S. provisional application serial No. 60/075,940, filed Feb. 25,1998, and from U.S. provisional application serial No. 60/077,033, filedMar. 6, 1998.

FIELD OF THE INVENTION

This invention relates to nucleic acids and encoded polypeptides whichinteract with TGF-β superfamily receptors and which are negativeregulators of signaling by those receptors. The invention also relatesto agents which bind the nucleic acids or polypeptides. The inventionfurther relates to methods of using such nucleic acids and polypeptidesin the treatment and/or diagnosis of disease.

BACKGROUND OF THE INVENTION

During mammalian embryogenesis and adult tissue homeostasis transforminggrowth factor β (TGF-β) performs pivotal tasks in intercellularcommunication (Roberts et al., 1993). The cellular effects of thispleiotropic factor are exerted by ligand-induced hetero-oligomerizationof two distantly related type I and type II serine/threonine kinasereceptors, TβR-I and TβR-II, respectively (Lin and Lodish, 1993;Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996).The two receptors, which both are required for signaling, act insequence; TβR-I is a substrate for the constitutively active TβR-IIkinase (Wrana et al., 1994; Weiser et al., 1995). TGF-β forms part of alarge family of structurally related proteins which include activins andbone morphogenetic proteins (BMPs) that signal in a similar fashion,each employing distinct complexes of type I and type II serine/threoninekinase receptors (Lin and Lodish, 1993; Derynck, 1994; Massague andWeis-Garcia, 1996; ten Dijke et al., 1996).

Genetic studies of TGF-β-like signalling pathways in Drosophila andCaenorhabditis elegans have led to the identification of mothers againstdpp (Mad) (Sekelsky et al., 1995) and sma (Savage et al., 1996) genes,respectively. The products of these related genes perform essentialfunctions downstream of TGF-β-like ligands acting via serine/threoninekinase receptors in these organisms (Wiersdorf et al, 1996; Newfeld etal., 1996; Hoodless et al., 1996). Vertebrate homologs of Mad and smahave been termed Smads (Derynck et al., 1996) or MADR genes (Wrana andAttisano, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have beenfound in specific tumor subsets, and thus Smads may function as tumorsuppressor genes (Hahn et al., 1996; Riggins et al., 1996; Eppert etal., 1996). Smad proteins share two regions of high similarity, termedMH1 and MH2 domains, connected with a variable proline-rich sequence(Massague, 1996; Derynck and Zhang, 1996). The C-terminal part of Smad2,when fused to a heterologous DNA-binding domain, was found to havetranscriptional activity (Liu et al., 1996; Meersseman et al., 1997).The intact Smad2 protein when fused to a DNA-binding domain, was latent,but transcriptional activity was unmasked after stimulation with ligand(Liu et al., 1996).

Different Smads specify different responses using functional assays inXenopus. Whereas, Smad1 induces ventral mesoderm, a BMP-like response,Smad2 induces dorsal mesoderm, an activin/TGF-β-like response (Graff etal., 1996; Baker and Harland, 1996; Thomsen, 1996). Upon ligandstimulation Smads become phosphorylated on serine and threonineresidues; BMP stimulates Smad1 phosphorylation, whereas TGF-β inducesSmad2 and Smad3 phosphorylation (Hoodless et al., 1996; Liu et al.,1996; Eppert et al., 1996; Lechleider et al., 1996; Yingling et al.,1996; Zhang et al., 1996; Macías-Silva et al., 1996; Nakao et al.,1996). Thus certain Smads are pathway specific. Pathway specific Smadsinclude Smad1, Smad2, Smad3 and Smad5.

Smad4 is a common component of TGF-β, activin and BMP signaling (Lagnaet al., 1996; Zhang et al., 1997; de Winter et al., 1997). Smad4phosphorylation has thus far been reported only after activinstimulation of transfected cells (Lagna et al., 1996). After stimulationwith TGF-β or activin Smad4 interacts with Smad2 or Smad3, and upon BMPchallenge a heteromeric complex of Smad4 and Smad1 has been observed(Lagna et al., 1996). Upon ligand stimulation, Smad complexestranslocate from the cytoplasm to the nucleus (Hoodless et al., 1996;Liu et al., 1996; Baker and Harland, 1996; Macías-Silva et al., 1996),where they, in combination with DNA-binding proteins, may regulate genetranscription (Chen et al., 1996).

SUMMARY OF THE INVENTION

The invention provides isolated Smad7 nucleic acid molecules, uniquefragments of those molecules, expression vectors containing theforegoing, and host cells transfected with those molecules. Theinvention also provides isolated polypeptides encoded by the Smad7nucleic acids and agents which bind such polypeptides, includingantibodies. The foregoing can be used in the diagnosis or treatment ofconditions characterized by the expression of a Smad7 nucleic acid orpolypeptide, or lack thereof. The invention also provides methods foridentifying pharmacological agents usefuil in the diagnosis or treatmentof such conditions. Here, we present the identification of Smad7, whichopposes pathway specific Smads including Smad1, Smad2 and Smad3 and thusis an inhibitor of the TGF-β superfamily signalling pathways.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The molecule hybridizes under stringent conditionsto a molecule consisting of the nucleic acid sequence of SEQ ID NOs:3 or5. The isolated nucleic acid molecule codes for a polypeptide whichinhibits TGF-β superfamily signaling. The invention further embracesnucleic acid lo molecules that differ from the foregoing isolatednucleic acid molecules in codon sequence due to the degeneracy of thegenetic code. The invention also embraces complements of the foregoingnucleic acids.

In certain embodiments, the isolated nucleic acid molecule comprises amolecule consisting of the nucleic acid sequence of SEQ ID NO:7 or 8.Preferably, the isolated nucleic acid molecule consists of the nucleicacid sequence of SEQ ID NO:7 or 8, or consists essentially of thenucleic acid sequence of SEQ ID NO:3 or 5.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule comprises amolecule consisting of a unique fragment of SEQ ID NO:3 between 12 and1944 nucleotides in length and complements thereof, or a unique fragmentof SEQ ID NO:5 between 12 and 1875 nucleotides in length and complementsthereof, provided that the isolated nucleic acid molecule excludessequences consisting only of SEQ ID NO:1 and SEQ ID NO:2. Preferably theisolated nucleic acid molecule excludes molecules consisting solely ofnucleotide sequences selected from the group consisting of accessionnumbers AA061644 (SEQ ID NO: 1), AA022262 (SEQ ID NO:2), AA347307,AA348247, 321995, W78627, W40869, AA033426, AA397050, AA016891, andC85115. In one embodiment, the isolated nucleic acid molecule consistsof between 12 and 32 contiguous nucleotides of SEQ ID NO:3, SEQ ID NO:5,or complements of such nucleic acid molecules. In preferred embodiments,the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguousnucleotides of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:8, or complements thereof.

According to another aspect of the invention, the invention involvesexpression vectors, and host cells transformed or transfected with suchexpression vectors, comprising the nucleic acid molecules describedabove.

According to still other aspects of the invention, transgenic non-humananimals are provided. The animals include in certain embodiments theforegoing expression vectors. In certain preferred embodiments, thetransgenic non-human animal includes a conditional Smad7 expressionvector, such as an expression vector that increases expression of Smad7in a tissue specific, development stage specific, or inducible manner.In other embodiments, the transgenic non-human animal has reducedexpression of Smad7 nucleic acid molecules. In some embodiments, thetransgenic non-human animal includes a Smad7 gene disrupted byhomologous recombination. The disruption can be homozygous orheterozygous. In other embodiments, the transgenic non-human animalincludes a conditional Smad7 gene disruption, such as one mediated bye.g. tissue specific, development stage specific, or inducible,expression of a recombinase. In yet other embodiments, the transgenicnon-human animal includes a transacting negative regulator of Smad7expression, such as antisense Smad7 nucleic acid molecules, nucleic acidmolecules which encode dominant negative Smad7 proteins, Smad7 directedribozymes, etc.

According to another aspect of the invention, an isolated polypeptide isprovided. The isolated polypeptide is encoded by the isolated nucleicacid molecule of any of claims 1, 2, 3, 4, 5 or 6, and the polypeptideinhibits TGF-β superfamily signaling activity.

In other embodiments, the isolated polypeptide consists of a fragment orvariant of the foregoing which retains the activity of the foregoing. Inpreferred embodiments, the fragment is a C-terminal fragment of Smad7,preferably amino acids 204-426 of SEQ ID NO:4 or SEQ ID NO:6, or aN-terminal fragment of Smad7, preferably amino acids 2-261 of SEQ IDNO:4 or SEQ ID NO:6.

According to another aspect of the invention, there are providedisolated polypeptides which selectively bind a Smad7 protein or fragmentthereof, provided that the isolated polypeptide is not a TGF-βsuperfamily type I receptor (e.g., a TGF-β, activin or BMP receptor).The isolated polypeptide in certain embodiments binds to a polypeptideencoded by the isolated. nucleic acid molecule of any of claims 1, 2, 3,4, 5 or 6. In preferred embodiments, isolated binding polypeptidesinclude antibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fdand antibody fragments which include a CDR3 region which bindsselectively to an epitope defined by the Smad7 polypeptides of theinvention, such as SEQ ID NOs:4 or 6). In other preferred embodiments,the polypeptide is an antibody or fragment thereof which selectivelybinds an epitope defined by a polypeptide selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14. In still other preferred embodiments, the isolated polypeptide isa monoclonal antibody, a humanized antibody or a chimeric antibody.

The invention provides in another aspect an isolated complex ofpolypeptides. The isolated complex includes a TGF-β superfamily receptorselected from the group consisting of activated TGFβ superfamily type Ireceptors and complexes of TGFβ superfamily type I receptors and TGFβsuperfamily type II receptors (e.g. a TGF-β, activin, Vg1 or BMPreceptors) bound to a polypeptide as claimed in claim 1. Preferably theisolated complex includes a polypeptide having the amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:6. In other preferred embodiments, thereceptor is selected from the group consisting of TβRI, BMPR-IA,BMPR-IB, ActR-IA, a complex of TβRI and TβRII, a complex of BMPR-IA andBMPR-II, a complex of BMPR-IB and BMPR-II, a complex of ActR-IA andBMPR-II and a complex of ActR-IA and ActR-II.

According to still another aspect of the invention, methods for reducingTGF-β superfamily signal transduction in a mammalian cell are provided.The methods involve contacting a mammalian cell with an amount of aninhibitor of TGF-β superfamily signal transduction effective to reducesuch signal transduction in the mammalian cell. Preferably the TGF-βsuperfamily signal transduction is mediated by a TGFβ superfamilyligand, particularly TGF-β1, activin, VG1, BMP-4 and/or BMP-7. Othermethods are provided for reducing phosphorylation of pathway specificSmads (e.g. Smad1, Smad2, Smad3 and/or Smad5) by contacting a mammaliancell with the inhibitor disclosed above. In certain embodiments of theforegoing methods, the inhibitor is an isolated Smad7 polypeptide or afragment thereof, such as a polypeptide encoded by a nucleic acid whichhybridizes under stringent conditions to SEQ ID NO:3 or 5, nucleic acidswhich encode the polypeptide of SEQ ID NO:4 or SEQ ID NO:6 ordegenerates or complements thereof. In some embodiments the nucleic acidencodes amino acids 204-426 of SEQ ID NO:4 or SEQ ID NO:6 or amino acids2-261 of SEQ ID NO:4 or SEQ ID NO:6. In still other embodiments, theinhibitor is an isolated Smad8 polypeptide or a fragment thereof.

According to still another aspect of the invention, methods formodulating proliferation and/or differentiation of a cell are provided.The methods involve contacting a cell with an amount of an isolatedSmad7 polypeptide,or a nucleic acid encoding and expressing such apolypeptide, as described above, effective to modulate the proliferationand/or differentiation of the cell.

The invention in a flrher aspect provides methods for increasing TGF-βsuperfamily signal transduction in a mammalian cell. The mammalian cellis contacted with an agent that selectively binds to an isolated nucleicacid molecule of the invention or an expression product thereof in anamount effective to increase TGF-β superfamily signal transduction.Preferably the TGF-β superfamily signal transduction is mediated by aTGFβ superfamily ligand selected from the group consisting of TGF-β1,activin, VG1, BMP-4 and BMP-7. Preferred agents are antisense nucleicacids, including modified nucleic acids, and polypeptides includingantibodies which bind to the polypeptide including the amino acids ofSEQ ID NO:4, the polypeptide including the amino acids of SEQ ID NO:6,the polypeptide including the amino acids of SEQ ID NO: 13, thepolypeptide including the amino acids of SEQ ID NO: 14, a N-terninalfragment of Smad7 or a C-terninal fragment of Smad7, and dominantnegative variants of the polypeptide of SEQ ID NO:4 or SEQ ID NO:6.

The invention in still another aspect provides compositions comprising aSmad7 polypeptide and a pharmaceutically acceptable carrier.

The invention in a further aspect involves methods for decreasing Smad7TGF-β superfamily inhibitory activity in a subject. An agent thatselectively binds to an isolated nucleic acid molecule of the inventionor an expression product thereof is administered to a subject in need ofsuch treatment, in an amount effective to decrease TGFβ superfamilysignal transduction inhibitory activity of Smad7 in the subject.Preferably the TGFβ superfamily signal transduction. is mediated by aTGFβ superfamily ligand selected from the group consisting of TGF-β1,activin, VG1, BMP-4 and BMP-7. Preferred agents are antisense nucleicacids, including modified nucleic acids, and polypeptides includingantibodies which bind to the polypeptide including the amino acids ofSEQ ID NO:4, the polypeptide including the amino acids of SEQ ID NO:6,the polypeptide including the amino acids of SEQ ID NO:13, thepolypeptide including the amino acids of SEQ ID NO:14, a N-terminalfragment of Smad7 or a C-terminal fragment of Smad7, and dominantnegative variants of the polypeptide of SEQ ID NO:4 or SEQ ID NO:6.

In another aspect the invention provides methods for diagnosinginduction of a TGF-β superfamily ligand in a cell. The methods includethe steps of (a) measuring the amount of Smad7 RNA or polypeptide in thecell and comparing the result of step (a) with a control.

According to still another aspect of the invention, methods are providedfor determining the presence of a functional TGFβ superfamily receptorin a cell. The methods include contacting the cell with an amount ofTGFβ superfamily ligand effective to increase the amount of Smad7 in thecell, measuring the amount of Smad7 RNA or polypeptide in the cell, andcomparing the result of the measurement with a control, wherein anincreased amount of Smad7 RNA or polypeptide in the cell indicates thepresence of a finctional TGFβ superfamily receptor. Preferably the TGFβsuperfamily receptor is selected from the group consisting of TGFβsuperfamily type I receptors, TGFβ superfamily type II receptors, andcomplexes of TGFβ superfamily type I receptors and TGFβ superfamily typeII receptors.

According to another aspect of the invention, methods are provided foridentifying lead compounds for a pharmacological agent useful in thediagnosis or treatment of disease associated with TGFβ superfamilysignal transduction inhibitory activity of Smad7. One set of methodsinvolves forming a mixture of a Smad7 polypeptide, a TGF-β superfamilyreceptor complex or an activated TGFβ superfamily type I receptor, and acandidate pharmacological agent. The mixture is incubated underconditions which, in the absence of the candidate pharmacological agent,permit a first amount of specific binding of the TGF-β superfamilyreceptor complex or activated TGFβ superfamily type I receptor by theSmad7 polypeptide. A test amount of the specific binding of the TGF-βsuperfamily receptor complex or activated type I receptor by the Smad7polypeptide then is detected. Detection of an increase in the foregoingactivity in the presence of the candidate pharmacological agentindicates that the candidate pharmacological agent is a lead compoundfor a pharmacological agent which increases the TGF-β superfamily signaltransduction inhibitory activity of Smad7. Detection of a decrease inthe foregoing activities in the presence of the candidatepharmacological agent indicates that the candidate pharmacological agentis a lead compound for a pharmacological agent which decreases the TGF-βsuperfamily signal transduction inhibitory activity of Smad7. Anotherset of methods involves forming a mixture as above, adding further apathway specific Smad polypeptide, and detecting first and test amountsof TGF-β superfamily induced phosphorylation of the pathway specificSmad polypeptide. Detection of an increase in the phosphorylation in thepresence of the candidate pharmacological agent indicates that thecandidate pharmacological agent is a lead compound for a pharmacologicalagent which decreases the TGF-β, superfamily signal transductioninhibitory activity of Smad7. Detection of a decrease in the foregoingactivities in the presence of the candidate pharmacological agentindicates that the candidate pharmacological agent is a lead compoundfor a pharmacological agent which increases the TGF-β, superfamilysignal transduction inhibitory activity of Smad7. Preferred Smad7polypeptides include the polypeptides of claim 18.

In the foregoing compositions and methods, preferred members of theTGF-β superfamily are TGF-β1, activin, VG1, BMP-4 and BMP-7, and thepreferred pathway specific Smad polypeptides are Smad1, Smad2, Smad3 andSmad5.

According to yet another aspect of the invention, a method for reducingexpression of a Smad6 or Smad7 nucleic acid or expression productthereof in a cell is provided. The method includes contacting the cellwith an amount of an agent which binds selectively to Smad4 effective toreduce the expression of the Smad6 or Smad7 nucleic acid or expressionproduct thereof in the cell. In certain embodiments, the agent is anantisense Smad4 molecule, or an antibody that selectively binds toSmad4.

In another aspect, the invention provides a method for increasing Smad6or Smad7 expression in a cell. The method includes contacting the cellwith an agent selected from the group consisting of activin, epidermalgrowth factor and phorbol esters in an amount effective to increaseSmad6 or Smad7 expression in the cell.

According to another aspect of the invention, a method for treating asubject having lung cancer characterized by elevated expression of aSmad6 gene or a Smad7 gene is provided. The method includesadministering to the subject an amount of an antisense nucleic acidwhich binds to the expression product of the Smad 6 or Smad7 geneeffective to reduce the expression of the Smad 6 or Smad7 gene. In otherembodiments, the method includes administering a polypeptide, such as anantibody or fragment thereof which binds a polypeptide selected from thegroup consisting of a polypeptide comprising the amino acid sequence ofSEQ ID NO:4, a polypeptide comprising the amino acid sequence of SEQ IDNO:6, a N-terminal fragment of Smad7 and a C-terminal fragment of Smad7,a polypeptide comprising the amino acid sequence of SEQ ID NO:10, apolypeptide comprising the amino acid sequence of SEQ ID NO:11, apolypeptide comprising the amino acid sequence of SEQ ID NO:12, apolypeptide comprising the amino acid sequence of SEQ ID NO:13, and apolypeptide comprising the amino acid sequence of SEQ ID NO:14. In stillother embodiments, the agent is a dominant negative variant of Smad6 orSmad7.

According to yet another aspect of the invention, methods for reducingeye defects in a developing mammalian embryo are provided. The methodsinclude contacting the cells of the embryo with an agent which reducesthe expression or activity of a Smad7 nucleic acid molecule or anexpression product thereof. In certain embodiments, the agentselectively binds the Smad7 nucleic acid molecule or an expressionproduct thereof. In preferred embodiments, the agent is an antisensenucleic acid molecule or a polypeptide, and preferably the polypeptideis an antibody or fragment thereof which binds a polypeptide selectedfrom the group consisting of a polypeptide comprising the amino acidsequence of SEQ ID NO:4, a polypeptide comprising the amino acidsequence of SEQ ID NO:6, a polypeptide comprising the amino acidsequence of SEQ ID NO:13, a polypeptide comprising the amino acidsequence of SEQ ID NO:14, a N-terminal fragment of Smad7 and aC-terminal fragment of Smad7. The agent also can be a dominant negativevariant of Smad7.

According to another aspect of the invention, an isolated polypeptide isprovided. The polypeptide includes a first polypeptide or fragmentthereof linked to a second polypeptide or fragment thereof, wherein thesecond polypeptide or fragment thereof comprises a MH2 domain of Smad7.The polypeptide is localized in the nucleus of a cell and is exportedfrom the nucleus to the cytoplasm of the cell upon TGFβ superfamilyreceptor-mediated signal transduction in cells having a TGFβ superfamilyreceptor.

According to another aspect of the invention, a fision protein isprovided which includes a Smad7 MH2 domain or a nuclear localizationfragment thereof. In other aspects of the invention, a fusion protein isprovided which includes a Smad7 MH2 domain or a transcriptionalactivation fragment thereof.

According to still another aspect of the invention, methods formodulating transcription of Smad7-regulated gene transcription areprovided. The methods include contacting a mammalian cell with an agentwhich modulates TGFβ superfamily receptor-mediated signal transductionin an amount effective to modulate Smad7-regulated gene transcription.In some embodiments the agent is a TGFβ superfamily ligand or aninhibitor of TGFβ superfamily receptor-mediated signal transduction.

In another aspect of the invention, an isolated nucleic acid molecule isprovided. The nucleic acid molecule includes the nucleotide sequence ofSEQ ID NO:15, an allelic variant thereof, or a functional fragmentthereof which confers TGFβ regulation. Nucleic acid molecules whichinclude the nucleotide sequences which hybridize under stringentconditions to SEQ ID NO:15 also are provided. In still other aspects ofthe invention, isolated nucleic acid molecules which include a uniquefragment of SEQ ID NO:15 are provided. Expression vector.s, whichinclude the foregoing isolated nucleic acid molecules including orrelated to SEQ ID NO:15 also are provided

According to another aspect of the invention, method for regulatingtranscription of a first nucleic acid molecule are provided. The methodsinclude preparing a construct comprising the first nucleic acid moleculeoperably linked to the foregoing isolated nucleic acid moleculesincluding or related to SEQ ID NO:15, and introducing the construct intoan expression system. In some embodiments the expression system is acell. In preferred embodiments, the cell expresses a TGFβ superfamilyreceptor, and the method includes contacting the cell with a TGFβsuperfamily ligand to increase expression of the first nucleic acidmolecule.

In still other aspects of the invention, methods for identifyingmodulators of TGFβ-regulated transcriptional activity are provided. Themethods include providing an expression system with a reporter constructincluding SEQ ID NO:15 or a TGFβ-regulated fragment thereof operablylinked to a nucleic acid encoding a detectable expression product, andcontacting the expression system with a candidate modulator compound.The expression system is incubated under conditions which, in theabsence of the candidate modulator, permit a first amount of expressionof the detectable expression product. A test amount of the expressionthe detectable expression product then is detected. Detection of anincrease in the foregoing activity in the presence of the candidatemodulator compound indicates that the candidate modulator compound is acompound which increases TGFβ-regulated transcriptional activity.Detection of a decrease in the foregoing activities in the presence ofthe candidate modulator compound indicates that the candidate modulatorcompound is a compound which decreases TGFβ-regulated transcriptionalactivity. In preferred embodiments the expression system is a cell or anin vitro transcription system. Other other preferred embodiments, thedetectable expression product is a reporter protein, such as an enzyme,e.g., luciferase, or a green fluorescent protein.

The use of the foregoing compositions in the preparation of a medicamentis also contemplated.

These and other aspects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict the cloning (human Smad7, SEQ ID NO:6; mouse Smad7,SEQ ID NO:4) and tissue distribution of Smad7.

FIGS. 2A-2I show that Smad7 inhibits TGF-β and activin inducedtranscriptional responses.

FIG. 3 demonstrates the association of Smad7 with the TGF-β receptorcomplex.

FIGS. 4A-4E show that Smad7 is not phosphorylated upon TβR-activation,but Smad7 overexpression inhibits TGF-β-mediated Smad2 and Smad3phosphorylation.

FIGS. 5A-5C show that Smad7 is an early response gene for TGF-β.

FIGS. 6A-6F demonstrate the association of Smad7 with BMP and activinreceptor complexes.

FIGS. 7A-7C show that Smad7 inhibits BMP- and activin-receptor mediatedphosphorylation of Smad1 or Smad5.

FIGS. 8A, 8B1, 8B2, 8C, 8D, and 8E demonstrates that the C-terminaldomain of Smad7 associates with the TGF-β seceptor complex and thatSmad8 and the N-terminal and C-terminal domains of Smad7 inhibit TGF-βsignalling.

FIGS. 9A-9C show a comparison of Smad6 and Smad7. A: comparison of theamino acid sequences. B: Pairwise alignment relationship between hSmad1through hSmad7 and hSmad9. C: Expression of Smad6 and Smad7 MnRNA inlung carcinoma cell lines.

FIGS. 10A1, 10A2, 10B, and 10C show that TGF-B superfamily membersinduce Smad6 and Smad7 mRNA levels.

FIG. 11 shows that anti-sense Smad7 mRNA potentiates TGF-β1transcriptional response.

FIGS. 12A-12B depict the effect of EGF and PMA on Smad6 and Smad7 mRNAexpression.

FIGS. 13A-13B show that Smad7 inhibits TGF-β-mediated signalingresponses more effectively than Smad8.

FIGS. 14A-14F show that overexpression of Smad7 in Xenopus embryosinduces formation of a partial secondary axis and eye defects.

FIG. 15 shows characterization of expression of Smad7 and Smad6 instable pMEP-4-driven transfected clones.

FIGS. 16A-16E show that Smad7 inhibits TGF-β1-mediated growth inhibition(A-C) Effective of Smad7 on TGF-β1-induced growth inhibition in threeindependent clones (D) Effective of Smad6S and (E) Smad6L onTGF-β1-induced growth inhibition.

FIGS. 17A-17F show that Smad7 inhibits TGF-β1-mediated induction ofearly response genes.

FIGS. 18A-18C show the subcellular distribution of Smad7 in the absenceor presence of TGF-β1 receptor activation.

FIGS. 19A-19E show the subcellular distribution of wild-type Smad7 andSmad7 deletion mutants in the absence or presence of TGF-β1 stimulation.

FIG. 20 depicts the results of a luciferase assays showing TGF-β1inducibility of a Smad7 promoter fragment.

FIG. 21 depicts several restriction enzyme sites and putative bindingsites for different anscription factors and Smad proteins in the ˜725 bpBam HI-Xho I minimal promoter fragment SEQ ID NO:15).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the mouse EST (accessionnumber A061644) which has sequence similarity to N-terminal Smaddomains.

SEQ ID NO:2 is the nucleotide sequence of the mouse EST (accessionnumber A022262) which has sequence similarity to C-terminal Smaddomains.

SEQ ID NO:3 is the nucleotide sequence of the mouse Smad7 cDNA.

SEQ ID NO:4 is the amino acid sequence of the mouse Smad7 protein.

SEQ ID NO:5 is the nucleotide sequence of the human Smad7 cDNA.

SEQ ID NO:6 is the amino acid sequence of the human Smad7 protein.

SEQ ID NO:7 is the nucleotide sequence of the coding region of the mouseSmad7 cDNA.

SEQ ID NO:8 is the nucleotide sequence of the coding region of the humanSmad7 cDNA.

SEQ ID NO:9 is the nucleotide sequence of the human Smad6 cDNA.

SEQ ID NO:10 is the amino acid sequence of the human Smad6 protein.

SEQ ID NO:11 is the amino acid sequence of a Smad7 preferred peptide towhich an antibody can be raised.

SEQ ID NO:12 is the amino acid sequence of a Smad7 preferred peptide towhich an antibody can be raised.

SEQ ID NO:13 is the amino acid sequence of a Smad7 preferred peptide towhich an antibody can be raised.

SEQ ID NO:14 is the amino acid sequence of a Smad7 preferred peptide towhich an antibody can be raised.

SEQ ID NO:15 is the 725 nucleotide Bam HI-Xho I Smad7 minimal promoterfragment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one aspect involves the cloning of a cDNAencoding a Smad7 TβR-I receptor-interacting protein. The sequence of themouse gene is presented as SEQ ID NO:3, and the predicted amino acidsequence of this gene's protein product is presented as SEQ ID NO:4. Thesequence of the human gene is presented as SEQ ID NO:5, and thepredicted amino acid sequence of this gene's protein product ispresented as SEQ ID NO:6. Analysis of the sequence by comparison tonucleic acid and protein databases determined that Smad7 has aC-terminal domain (the MH2 domain) which is related to other Smadproteins. The Smad7 C-terminal domain is most related to Smad6 (48%identity).

The invention thus involves in one aspect Smad7 polypeptides, genesencoding those polypeptides, finctional modifications and variants ofthe foregoing, useful fragments of the foregoing, as well astherapeutics relating thereto. The expression of these genes affects andis affected by TGF-β superfamily expression and signal transduction. TheTGF-β superfamily members are well known to those of ordinary skill inthe art and include TGF-βs, activins, bone morphogenetic proteins(BMPs), VG1, Mullerian inhibitory substance (MIS) andgrowth/differentiation factors (GDFs).

Homologs and alleles of the Smad7 nucleic acids of the invention can beidentified by conventional techniques. Thus, an aspect of the inventionis those nucleic acid sequences which code for Smad7 polypeptides andwhich hybridize to a nucleic acid molecule consisting of the codingregion of SEQ ID NO:3 or SEQ ID NO:5, under stringent conditions. Theterm “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references which compile such methods, e.g. Molecular Cloning:A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDSis sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS attemperatures up to 65° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of Smad7 nucleic acids of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

In general homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to SEQ IDNOs:3 or 5 and SEQ ID NOs:4 or 6, respectively, in some instances willshare at least 50% nucleotide identity and/or at least 65% amino acididentity and in still other instances will share at least 60% nucleotideidentity and/or at least 75% amino acid identity. Watson-Crickcomplements of the foregoing nucleic acids also are embraced by theinvention.

In screening for Smad7 proteins, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is fmally transferred, themembrane can be placed against X-ray film to detect the radioactivesignal.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating Smad7polypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, COT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

The invention also provides isolated unique fragments of SEQ ID NOs:3 or5 or complements of SEQ ID NOs:3 or 5. A unique fragment is one that isa ‘signature’ for the larger nucleic acid. It, for example, is longenough to assure that its precise sequence is not found in moleculesoutside of the Smad7 nucleic acids defined above. A unique fragmentexcludes, by definition, sequences consisting solely of EST sequencessuch as those described by SEQ ID NOs:1 and 2. Unique fragments can beused as probes in Southern blot assays to identify such nucleic acids,or can be used in amplification assays such as those employing PCR. Asknown to those skilled in the art, large probes such as 200 nucleotidesor more are preferred for certain uses such as Southern blots, whilesmaller fragments will be preferred for uses such as PCR. Uniquefragments also can be used to produce fusion proteins for generatingantibodies or determining binding of the polypeptide fragments, asdemonstrated in the Examples, or for generating immunoassay components.Likewise, unique fragments can be employed to produce nonfused fragmentsof the Smad7 polypeptides such as the N-terminal and C-terminalfragments disclosed herein, useful, for example, in the preparation ofantibodies, in immunoassays, and as a competitive binding partner of theTGF-β, activin, BMP receptors and/or other polypeptides which bind tothe Smad7 polypeptides, for example, in therapeutic applications. Uniquefragments further can be used as antisense molecules to inhibit theexpression of Smad7 nucleic acids and polypeptides, particularly fortherapeutic purposes as described in greater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:3 and/or SEQ ID NO:5 and its complementwill require longer segments to be unique while others will require onlyshort segments, typically between 12 and 32 nucleotides (e.g. 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31and 32 bases long). Virtually any segment of SEQ ID NO:7 or SEQ ID NO:8,or complements thereof, that is 18 or more nucleotides in length will beunique. Those skilled in the art are well versed in methods forselecting such sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-Smad7 nucleic acids. A comparison of the sequence of the fragment tothose on known data bases typically is all that is necessary, althoughin vitro confirmatory hybridization and sequencing analysis may beperformed.

A unique fragment can be a functional fragment. A functional fragment ofa nucleic acid molecule of the invention is a fragment which retainssome functional property of the larger nucleic acid molecule, such ascoding for a finctional polypeptide, binding to proteins, regulatingtranscription of operably linked nucleic acids, and the like. One ofordinary skill in the art can readily determine using the assaysdescribed herein and those well known in the art tc determine whether afragment is a functional fragment of a nucleic acid molecule using nomore than routine experimentation.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a Smad7polypeptide, to increase TGF-β, activin and/or BMP signaling by reducingthe amount of Smad7. This is desirable in virtually any medicalcondition wherein a reduction of Smad7 is desirable, e.g., to increaseTGF-β signaling.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NOs:3, 5 or 9, or upon allelic orhomologous genomic and/or cDNA sequences, one of skill in the art caneasily choose and synthesize any of a number of appropriate antisensemolecules for use in accordance with the present invention. In order tobe sufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably,the antisense oligonucleotides comprise a complementary sequence of20-30 bases. Although oligonucleotides may be chosen which are antisenseto any region of the gene or mRNA transcripts, in preferred embodimentsthe antisense oligonucleotides correspond to N-terminal or 5′ upstreamsites such as translation initiation, transcription initiation orpromoter sites. In addition, 3′-untranslated regions may be targeted.Targeting to mRNA splicing sites has also been used in the art but maybe less preferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439-457, 1994) and at which proteins are not expectedto bind. Finally, although SEQ ID Nos:3, 5 and 9 disclose cDNAsequences, one of ordinary skill in the art may easily derive thegenomic DNA corresponding to the cDNA of SEQ ID Nos:3, 5 or 9. Thus, thepresent invention also provides for antisense oligonucleotides which arecomplementary to the genomic DNA corresponding to SEQ ID Nos:3, 5 and 9.Similarly, antisense to allelic or homologous cDNAs and genomic DNAs areenabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterintemucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic intemucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic intemucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisens,e molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding Smad7polypeptides, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding Smad7 polypeptide or fragment or variantthereof. That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J Cancer, 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also permits the construction of Smad7 gene “knock-outs”in cells and in animals, providing materials for studying certainaspects of TGF-β, activin and/or BMP signal transduction.

The invention also provides isolated polypeptides, which include thepolypeptides of SEQ ID NOs:4 and 6 and unique fragments of SEQ ID NOs:4and 6 including fragments comprising amino acids 2-261 of SEQ ID NOs:4and 6 and amino acids 204-426 of SEQ ID NOs:4 and 6. Such polypeptidesare useful, for example, alone or as fusion proteins to generateantibodies, as a components of an immunoassay.

A unique fragment of an Smad7 polypeptide, in general, has the featuresand characteristics of unique fragments as discussed above in connectionwith nucleic acids. As will be recognized by those skilled in the art,the size of the unique fragment will depend upon factors such as whetherthe fragment constitutes a portion of a conserved protein domain. Thus,some regions of amino acids 2-261 of SEQ ID NOs:4 and 6 and amino acids204-426 of SEQ ID NOs:4 and 6 will require longer segments to be uniquewhile others will require only short segments, typically between 5 and12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long).Virtually any segment of amino acids 2-261 of SEQ ID NOs:4 and 6 andamino acids 204-426 of SEQ ID NOs:4 and 6, that is 10 or more aminoacids in length will be unique.

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptides(such as TβR-I) or fragments thereof, selective binding of nucleic acidsor proteins, and enzymatic activity. For example, as exemplified herein,N-terminal and C-terminal Smad7 fragments such as those which includesamino acids 2-261 or 204-426 of SEQ ID NO:4 or SEQ ID NO:6 can be usedas a finctional equivalent of full length Smad7 in the methods of theinvention, including e.g., inhibition of TGF-β signal transduction.Other Smad polypeptide fragments, e.g., other N-terminal or C-terminalfragments, can be selected according to their functional properties. Forexample, one of ordinary skill in the art can prepare Smad7 fragmentsrecombinantly and test those fragments according to the methodsexemplified below, such as binding to a TGFβ superfamily receptor, orinhibition of pathway specific Smad polypeptide phosphorylation. Thoseskilled in the art also are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known data bases typically is all that is necessary.

The invention embraces variants of the Smad7 polypeptides describedabove. As used herein, a “variant” of a Smad7 polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a Smad7 polypeptide. Modifications which create aSmad7 variant can be made to a Smad7 polypeptide 1) to reduce oreliminate an activity of a Smad7 polypeptide, such as binding to TβR-I;2) to enhance a property of a Smad7 polypeptide, such as proteinstability in an expression system or the stability of protein-proteinbinding; or 3) to provide a novel activity or property to a Smad7polypeptide, such as addition of an antigenic epitope or addition of adetectable moiety. Modifications to a Smad7 polypeptide are typicallymade to the nucleic acid which encodes the Smad7 polypeptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids or non-amino acid moieties.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the Smad7 amino acid sequence.

In general, variants include Smad7 polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aSmad7 polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a Smad7 polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such a hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant Smad7 polypeptides) which are silent asto the amino acid sequence of the polypeptide, but which providepreferred codons for translation in a particular host. The preferredcodons for translation of a nucleic acid in, e.g., E. coli, are wellknown to those of ordinary skill in the art. Still other mutations canbe made to the noncoding sequences of a Smad7 gene or cDNA clone toenhance expression of the polypeptide. The activity of variants of Smad7polypeptides can be tested by cloning the gene encoding the variantSmad7 polypeptide into a bacterial or mammalian expression vector,introducing the vector into an appropriate host cell, expressing thevariant Smad7 polypeptide, and testing for a functional capability ofthe Smad7 polypeptides as disclosed herein. For example, the variantSmad7 polypeptide can be tested for inhibition of TβR-I, activin and/orBMP receptor signaling activity as disclosed in the Examples, or forinhibition of Smad1, Smad2, Smad3 and/or Smad5 phosphorylation as isalso disclosed herein. Preparation of other variant polypeptides mayfavor testing of other activities, as will be known to one of ordinaryskill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in Smad7 polypeptides to provide functionallyequivalent variants of the foregoing polypeptides, i.e, the variantsretain the functional capabilities of the Smad7 polypeptides. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants of the Smad7 polypeptides include conservative amino acidsubstitutions of SEQ ID NOs:4 or 6. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Conservative amino-acid substitutions in the amino acid sequence ofSmad7 polypeptides to produce functionally equivalent variants of Smad7polypeptides typically are made by alteration of a nucleic acid encodinga Smad7 polypeptide (SEQ ID NOs:3 and 5). Such substitutions can be madeby a variety of methods known to one of ordinary skill in the art. Forexample, amino acid substitutions may be made by PCR-directed mutation,site-directed mutagenesis according to the method of Kunkel (Kunkel,Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemicalsynthesis of a gene encoding a Smad7 polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a Smad7polypeptide, such as a TβR-I binding site peptide, the substitutions canbe made by directly synthesizing the peptide. The activity offunctionally equivalent fragments of Smad7 polypeptides can be tested bycloning the gene encoding the altered Smad7 polypeptide into a bacterialor mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered Smad7 polypeptide, andtesting for a functional capability of the Smad7 polypeptides asdisclosed herein. Peptides which are chemically synthesized can betested directly for function, e.g., for binding to TβR-I, ActR-IB and/orBMPR-IB.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the Smad7 protein molecules (SEQ ID NOs:4 and 6). A variety ofmethodologies well-known to the skilled practitioner can be utilized toobtain isolated Smad7 molecules. The polypeptide may be purified fromcells which naturally produce the polypeptide by chromatographic meansor imununological recognition. Alternatively, an expression vector maybe introduced into cells to cause production of the polypeptide. Inanother method, mRNA transcripts may be microinjected or otherwiseintroduced into cells to cause production of the encoded polypeptide.Translation of mRNA in cell-free extracts such as the reticulocytelysate system also may be used to produce polypeptide. Those skilled inthe art also can readily follow known methods for isolating Smad7polypeptides. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

The isolation of the Smad7 gene also makes it possible for the artisanto diagnose a disorder characterized by expression of Smad7. Thesemethods involve determining expression of the Smad7 gene, and/or Smad7polypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction asexemplified in the examples below, or assaying with labeledhybridization probes.

The invention also makes it possible isolate proteins such as TβR-I,ActR-IB and BMPR-IB by the binding of such proteins to Smad7 asdisclosed herein. The identification of this binding also permits one ofskill in the art to block the binding of Smad7 to other proteins, suchas TβR-I, as well as blocking the binding of other Smads, such as Smad2or Smad3 to TβR-I receptors. Binding of the proteins can be effected byintroducing into a biological system in which the proteins bind (e.g., acell) a polypeptide including a Smad7 TβR-I binding site in an amountsufficient to block the binding. The identification of a TβR-I domainbinding site in Smad7 also enables one of skill in the art to preparemodified proteins, using standard recombinant DNA techniques, which canbind to proteins such as TβR-I, ActR-IB and as BMPR-IB. For example,when one desires to target a certain protein to a TGF-β receptorcomplex, one can prepare a fusion polypeptide of the protein and theSmad7 TβR-I binding site. Additional uses are described further herein.

The invention further provides methods for reducing or increasing TGF-βfamily signal transduction in a cell. Such methods are useful in vitrofor altering the TGF-β signal transduction, for example, in testingcompounds for potential to block aberrant TGF-β signal transduction orincrease deficient TGF-β signal transduction. In vivo, such methods areuseful for modulating growth, e.g., to treat cancer and fibrosis.Increasing TGF-β signal transduction in a cell by, e.g., introducing adominant negative Smad7 polypeptide or Smad7 antisense oligonucleotidesin the cell, can be used to provide a model system for testing theeffects of putative inhibitors of TGF-β signal transduction. Suchmethods also are useful in the treatment of conditions which result fromexcessive or deficient TGF-β signal transduction. TGF-β signaltransduction can be measured by a variety of ways known to one ofordinary skill in the art, such as the reporter systems described in theExamples. Various modulators of Smad7 activity can be screened foreffects on TGF-β signal transduction using the methods disclosed herein.The skilled artisan can first determine the modulation of a Smad7activity, such as TGF-β signaling activity, and then apply such amodulator to a target cell or subject and assess the effect on thetarget cell or subject. For example, in screening for modulators ofSmad7 useful in the treatment of cancer, cells in culture can becontacted with Smad7 modulators and the increase or decrease of growthor focus formation of the cells can be determined according to standardprocedures. Smad7 activity modulators can be assessed for their effectson other TGF-β signal transduction downstream effects by similar methodsin many cell types. The foregoing also applies to signalling via activinand BMP complexes.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from SEQ ID NOs:4 and/or 6. A dominant negativepolypeptide is an inactive variant of a protein, which, by interactingwith the cellular machinery, displaces an active protein from itsinteraction with the cellular machinery or competes with the activeprotein, thereby reducing the effect of the active protein. For example,a dominant negative receptor which binds a ligand but does not transmita signal in response to binding of the ligand can reduce the biologicaleffect of expression of the ligand. Likewise, a dominant negativecatalytically-inactive kinase which interacts normally with targetproteins but does not phosphorylate the target proteins can reducephosphorylation of the target proteins in response to a cellular signal.Similarly, a dominant negative transcription factor which binds to apromoter site in the control region of a gene but does not increase genetranscription can reduce the effect of a normal transcription factor byoccupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,given the teachings contained herein of a Smad7 polypeptide, one ofordinary skill in the art can modify the sequence of the Smad7polypeptide by site-specific mutagenesis, scanning mutagenesis, partialgene deletion or truncation, and the like. See, e.g., U.S. Pat. No.5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilledartisan then can test the population of mutagenized polypeptides fordiminution in a selected activity (e.g., Smad7 reduction of TGF-βsignalling activity) and/or for retention of such an activity. Othersimilar methods for creating and testing dominant negative variants of aprotein will be apparent to one of ordinary skill in the art.

Dominant negative Smad7 proteins include variants in which a portion ofthe TβR-I, activin receptor or BMP receptor binding site has beenmutated or deleted to reduce or eliminate Smad7 interaction with theTGF-β, activin, or BMP receptor complex respectively. Other examplesinclude Smad7 variants in which the ability to inhibit phosphorylationof Smad2 and/or Smad3 is reduced. One of ordinary skill in the art canreadily prepare and test Smad7 variants bearing mutations or deletionsin the C-terminal domain (e.g., in the MH2 domain) or in the N-terminaldomain (e.g., in the glycine/glutamic acid residue rich region).

The invention also involves agents such as polypeptides which bind toSmad7 polypeptides and to complexes of Smad7 polypeptides and bindingpartners such as TβR-I, ActR-IB and BMPR-IB. Such binding agents can beused, for example, in screening assays to detect the presence or absenceof Smad7 polypeptides and complexes of Smad7 polypeptides and theirbinding partners and in purification protocols to isolate Smad7polypeptides and complexes of Smad7 polypeptides and their bindingpartners. Such agents also can be used to inhibit the native activity ofthe Smad7 polypeptides or their binding partners, for example, bybinding to such polypeptides, or their binding partners or both.

The invention, therefore, embraces peptide binding agents which, forexample, can be antibodies or fragments of antibodies having the abilityto selectively bind to Smad7 polypeptides. Antibodies include polyclonaland monoclonal antibodies, prepared according to conventionalmethodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modem Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from whictL the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to Smad7 polypeptides, and complexes of both Smad7polypeptides and their binding partners. These polypeptides may bederived also from sources other than antibody technology. For example,such polypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilized formor as phage display libraries. Combinatorial libraries also can besynthesized of peptides containing one or more amino acids. Librariesfurther can be synthesized of peptoids and non-peptide syntheticmoieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe Smad7 polypeptide. This process can be repeated through severalcycles of reselection of phage that bind to the Smad7 polypeptide.Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds to the Smad7 polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to the Smad7 polypeptides. Thus, the Smad7 polypeptides of theinvention, or a fragment thereof, can be used to screen peptidelibraries, including phage display libraries, to identify and selectpeptide binding partners of the Smad7 polypeptides of the invention.Such molecules can be used, as described, for screening assays, forpurification protocols, for interfering directly with the functioning ofSmad7 and for other purposes that will be apparent to those of ordinaryskill in the art.

A Smad7 polypeptide, or a fragment thereof, also can be used to isolatetheir native binding partners, including, e.g., the TGF-β, activin, orBMP receptor complexes. Isolation of such binding partners may beperformed according to well-known methods. For example, isolated Smad7polypeptides can be attached to a substrate (e.g., chromatographicmedia, such as polystyrene beads, or a filter), and then a solutionsuspected of containing the TGF-β receptor complex may be applied to thesubstrate. If a TGF-β receptor complex which can interact with Smad7polypeptides is present in the solution, then it will bind to thesubstrate-bound Smad7 polypeptide. The TGF-β receptor complex then maybe isolated. Other proteins which are binding partners for Smad7, suchas other Smads, activin receptor complexes, and BMP receptor complexesmay be isolated by similar methods without undue experimentation.

It will also be recognized that the invention embraces the use of theSmad7 cDNA sequences in expression vectors, as well as to transfect hostcells and cell lines, be these prokaryotic (e.g., E. coli), oreukaryotic (e.g., CHO cells, COS cells, yeast expression systems andrecombinant baculovirus expression in insect cells). Especially usefulare mammalian cells such as human, mouse, hamster, pig, goat, primate,etc. They may be of a wide variety of tissue types, and include primarycells and cell lines. Specific examples include keratinocytes,peripheral blood leukocytes, bone marrow stem cells and embryonic stemcells. The expression vectors require that the pertinent sequence, i.e.,those nucleic acids described supra, be operably linked to a promoter.

The invention also includes transgenic non-human animals. As usedherein, “transgenic non-human animals” includes non-human animals havingone or more exogenous nucleic acid molecules incorporated in germ linecells and/or somatic cells. Thus the transgenic animal include“knockout” animals having a homozygous or heterozygous gene disruptionby homologous recombination, animals having episomal or chromosomallyincorporated expression vectors, etc. Knockout animals can be preparedby homologous recombination using embryonic stem cells as is well knownin the art. The recombination can be facilitated by the cre/lox systemor other recombinase systems known to one of ordinary skill in the art.In certain embodiments, the recombinase system itself is expressedconditionally, for example, in certain tissues or cell types, at certainembryonic or post-embryonic developmental stages, inducibly by theaddition of a compound which increases or decreases expression, and thelike. In general, the conditional expression vectors used in suchsystems use a variety of promoters which confer the desired geneexpression pattern (e.g., temporal or spatial). Conditional promotersalso can be operably linked to Smad7 nucleic acid molecules to increaseexpression of Smad7 in a regulated or conditional manner. Trans-actingnegative regulators of Smad7 activity or expression also can be operablylinked to a conditional promoter as described above. Such trans-actingregulators include antisense Smad7 nucleic acids molecules, nucleic acidmolecules which encode dominant negative Smad7 molecules, ribozymemolecules specific for Smad7 nucleic acids, and the like. The transgenicnon-human animals are useful in experiments directed toward testingbiochemical or physiological effects of diagnostics or therapeutics forconditions characterized by increased or decreased Smad7 expression.Other uses will be apparent to one of ordinary skill in the art.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents active at the levelof a Smad7 or Smad7 fragment modulatable cellular function. Inparticular, such flnctions include TGF-β, activin and/or BMP signaltransduction and formation of a TGF-β, activin and/or BMP receptor-Smad7protein complex. Generally, the screening methods involve assaying forcompounds which interfere with a Smad7 activity such as TGF-βreceptor-Smad7 binding, etc, although compounds which enhance Smad7activity also can be assayed using the screening methods. Such methodsare adaptable to automated, high throughput screening of compounds. Thetarget therapeutic indications for pharmacological agents detected bythe screening methods are limited only in that the target cellularfunction be subject to modulation by alteration of the formation of acomplex comprising a Smad7 polypeptide or fragment thereof and one ormore natural Smad7 intracellular binding targets, such as TGF-βreceptor. Target indications include cellular processes modulated byTGF-β, activin and/or BMP signal transduction following receptor-ligandbinding.

A wide variety of assays for pharmacological agents are provided,including, labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays, cell-based assayssuch as two- or three-hybrid screens, expression assays, etc. Forexample, three-hybrid screens are used to rapidly examine the effect oftransfected nucleic acids on the intracellular binding of Smad7 or Smad7fragments to specific intracellular targets. The transfected nucleicacids can encode, for example, combinatorial peptide libraries orantisense molecules. Convenient reagents for such assays, e.g., GAL4fusion proteins, are known in the art. An exemplary cell-based assayinvolves transfecting a cell with a nucleic acid encoding a Smad7polypeptide fused to a GAL4 DNA binding domain and a nucleic acidencoding a TGF-β receptor domain which interacts with Smad7 fused to atranscription activation domain such as VP16. The cell also contains areporter gene operably linked to a gene expression regulatory region,such as one or more GAL4 binding sites. Activation of reporter genetranscription occurs when the Smad7 and TGF-β receptor fusionpolypeptides bind such that the GAL4 DNA binding domain and the VP16transcriptional activation domain are brought into proximity to enabletranscription of the reporter gene. Agents which modulate a Smad7polypeptide mediated cell function are then detected through a change inthe expression of reporter gene. Methods for determining changes in theexpression of a reporter gene are known in the art.

Smad7 fragments used in the methods, when not produced by a transfectednucleic acid are added to an assay mixture as an isolated polypeptide.Smad7 polypeptides preferably are produced recombinantly, although suchpolypeptides may be isolated from biological extracts. Recombinantlyproduced Smad7 polypeptides include chimeric proteins comprising afusion of a Smad7 protein with another polypeptide, e.g., a polypeptidecapable of providing or enhancing protein-protein binding, sequencespecific nucleic acid binding (such as GAL4), enhancing stability of theSmad7 polypeptide under assay conditions, or providing a detectablemoiety, such as green fluorescent protein or Flag epitope as provided inthe examples below.

The assay mixture is comprised of a natural intracellular Smad7 bindingtarget such as a TGF-β receptor or fragment thereof capable ofinteracting with Smad7. While natural Smad7 binding targets may be used,it is frequently preferred to use portions (e.g., peptides or nucleicacid fragments) or analogs (i.e., agents which mimic the Smad7 bindingproperties of the natural binding target for purposes of the assay) ofthe Smad7 binding target so long as the portion or analog providesbinding affinity and avidity to the Smad7 fragment measurable in theassay.

The assay mixture also comprises a candidate pharmacological agent.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate pharmacological agents aresmall organic compounds, i.e., those having a molecular weight of morethan 50 yet less than about 2500, preferably less than about 1000 and,more preferably, less than about 500. Candidate agents comprisefunctional chemical groups necessary for structural interactions withpolypeptides and/or nucleic acids, and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups and more preferably at least three of thefinctional chemical groups. The candidate agents can comprise cycliccarbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfinctional groups. Candidate agents also can be biomolecules such aspeptides, saccharides, fatty acids, sterols, isoprenoids, purines,pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the agent is a nucleic acid,the agent typically is a DNA or RNA molecule, although modified nucleicacids as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease, inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, the Smad7 polypeptide specifically binds thecellular binding target, a portion thereof or analog thereof. The orderof addition of components, incubation temperature, time of incubation,and other perimeters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe Smad7 polypeptide and one or more binding targets is detected by anyconvenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximum signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of Smad7 polypeptide interacting witha target molecule typically encodes a directly or indirectly detectableproduct, e.g., β-galactosidase activity, luciferase activity, and thelike. For cell free binding assays, one of the components usuallycomprises, or is coupled to, a detectable label. A wide variety oflabels can be used, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc). orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseseradish peroxidase, etc.). The label may be bound to aSmad7 binding partner, or incorporated into the structure of the bindingpartner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art.

The invention provides Smad7-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, Smad7-specificpharmacological agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with improper utilization of a pathway involving Smad7,e.g., TGF-β induced phosphorylation of Smad2 or Smad3, TGF-βreceptor-Smad7 complex formation, activin or BMP signalling, etc. NovelSmad7-specific binding agents include Smad7-specific antibodies andother natural intracellular binding agents identified with assays suchas two hybrid screens, and non-natural intracellular binding agentsidentified in screens of chemical libraries and the like.

In general, the specificity of Smad7 binding to a binding agent is shownby binding equilibrium constants. Targets which are capable ofselectively binding a Smad7 polypeptide preferably have bindingequilibrium constants of at least about 10⁷ M⁻¹, more preferably atleast about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. Thewide variety of cell based and cell free assays may be used todemonstrate Smad7-specific binding. Cell based assays include one, twoand three hybrid screens, assays in which Smad7-mediated transcriptionis inhibited or increased, etc. Cell free assays include Smad7-proteinbinding assays, immunoassays, etc. Other assays useful for screeningagents which bind Smad7 polypeptides include fluorescence resonanceenergy transfer (FRET), and electrophoretic mobility shift analysis(EMSA).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a retrovirus, or othervirus; a liposome) can have a targeting molecule attached thereto. Forexample, a molecule such as an antibody specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellcan be bound to or incorporated within the nucleic acid deliveryvehicle. For example, where liposomes are employed to deliver thenucleic acids of the invention, proteins which bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a composition that alone, ortogether with further doses, produces the desired response, e.g. altersfavorably the signal transduction resulting from binding of TGF-β,activin, BMP and/or Vg1 (Vgr-1) to specific receptors. In the case oftreating a particular disease, such as cancer, the desired response isinhibiting the progression of the disease. This may involve only slowingthe progression of the disease temporarily, although more preferably, itinvolves halting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practioner. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is generally preferred that amaximum dose of the individual components or combinations thereof beused, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of Smad7 or nucleic acidencoding Smad7 for producing the desired response in a unit of weight orvolume suitable for administration to a patient. The response can, forexample, be measured by determining the signal transduction enhanced orinhibited by the Smad7 composition via a reporter system as describedherein, by measuring downstream effects such as gene expression, or bymeasuring the physiological effects of the Smad7 composition, such asregression of a tumor or decrease of disease symptoms. Likewise, theeffects of antisense Smad6 and Smad7 can be readily determined bymeasuring expression of the individual genes in cells to which anantisense composition is added. Other assays will be known to one ofordinary skill in the art and can be employed for measuring the level ofthe response.

The doses of Smad7 polypeptide or nucleic acid administered to a subjectcan be chosen in accordance with different parameters, in particular inaccordance with the mode of administration used and the state of thesubject. Other factors include the desired period of treatment. In theevent that a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits.

In general, doses of Smad7 are formulated and administered in dosesbetween 1 ng and 1 mg, and preferably between 10 ng and 100 μg,according to any standard procedure in the art. Where nucleic acidsencoding Smad7 of variants thereof are employed, doses of between 1 ngand 0.1 mg generally will be formulated and administered according tostandard procedures. Other protocols for the administration of Smad7compositions will be known to one of ordinary skill in the art, in whichthe dose amount, schedule of injections, sites of injections, mode ofadministration (e.g., intra-tumoral) and the like vary from theforegoing. Administration of Smad7 compositions to mammals other thanhumans, e.g. for testing purposes or veterinary therapeutic purposes, iscarried out under substantially the same conditions as described above.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

Smad7 may be combined, if desired, with a pharmaceutically-acceptablecarrier. The term “pharmaceutically-acceptable carrier” as used hereinmeans one or more compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of Smad7polypeptides or nucleic acids, which is preferably isotonic with theblood of the recipient. This preparation may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono-or di-glycerides. In addition, fatty acids suchas oleic acid may be used in the preparation of injectables. Carrierformulation suitable for oral, subcutaneous, intravenous, intramuscular,etc. administrations can be found in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.

In another aspect of the invention, Smad7 polypeptides or nucleic acidare used in the manufacture of a medicament for modulating a TGF-β,activin, BMP or Vg1 response. The medicament can be placed in a vial andbe incorporated into a kit to be used for increasing a subject'sresponse to one or more of the above TGF-β family members. In certainembodiments, other medicaments which modulate the same responses orwhich favorably affect the Smad7 compositions can also be included inthe same kit. The kits can include instructions or other printedmaterial on how to administer the Smad7 compositions and any othercomponents of the kit.

EXAMPLES Methods

Isolation of mSmad7 and hSmad7 cDNA and Northern Blot Analysis.

cDNA encoding the complete mSmad7 was made by fusing mouse EST cDNA(AA061644) with a partial cDNA isolated from a mouse placenta library.The cDNA for hSmad7 was isolated by screening a human brain cDNAlibrary. cDNAs were sequenced on both strands using an ABI310 GeneticAnalyzer. Isolation of total RNA, Northern blot analysis was performedas described before (Afrakhte, M. et al., Int. J. Cancer 68, 802-809(1996) using QUIKHYB® hybridization solution from Stratagene (La Jolla,Calif.).

Expression Plasmids.

Expression constructs for TβR-I, TβR-II, BMPR-IA, BMPR-IB, ActR-I,ActR-IB, Smad1, Smad2, Smad3 and Smad5 have been described previously(Nakao et al., EMBO J. 16:5353-5362, 1997). Other Smad or receptorplasmids have been previously described or were prepared according tostandard protocols. F-Smad7 and F-Smad8 were made by a PCR-directedapproach and subcloning into pcDNA-Flag. N-terminal and C-termninalSmad7 expression vectors were prepared using PCR amplification withspecific primers and Smad7 cDNA as template. Expression constructs forthe C-terminal domain of F-Smad7 (7C; amino acids 204-426), F-Smad7Cwith C-tail deletion (7CΔ; amino acids 204-207), Smad7 with deletion ofC-tail (7Δ; amino acids 1-407), the N-terminal domain of Smad7 (7N;amino acids 1-203), mouse F-Smad6 “long version” (F-Smad6L)(Imamura etal., 1997) and human F-Smad6 “short version” (F-Smad6S) (Topper et al.,1997), were made by a PCR-directed approach and were subcloned intopcDNA3-Flag. The resulting expression plasmids expressed N-terminal andC-terminal Smad domains tagged at their N-termini with the Flag epitope.Alternatively, the Smads and Smad fragments were subcloned into pMEP4for inducible expression of the proteins. Anti-sense expressionconstructs for Smad molecules including mSmad7 and hSmad3 were preparedby cloning the complete Smad coding regions in reverse direction inpcDNA3 expression vector (Invitrogen, Carlsbad, Calif.).

Cell Assays.

Transient transfection, metabolic labeling, immunoprecipitation,[³²P]orthophosphate labeling of cells, and SDS-PAGE were performed asdescribed previously (Nakao et al., 1997).

Iodination of Ligand and Affinity Cross-linking.

Iodination of TGF-β1 and affinity cross-linking followed byimmunoprecipitation were performed as previously described (Nakao etal., 1997). TGF-β1 and BMP-7 were iodinated using the chloramine-Tmethod according to Frolik et al (J. Biol. Chem. 259:10995-11000, 1984).Cross-linking was performed as previously described (Nakao et al.,1997). In some cases, incubation with ¹²⁵I-TGF-β1 was performed at roomtemperature for 2 h (Souchelnytskyi et al., J. Biol. Chem.272:27678-27685, 1997) and the transfected cells lines were stimulatedfor 24 h with 100 μM/ml zinc chloride to induce the expression of Smad7.Complexes of Smads and affinity-labeled receptors wereimmunoprecipitated with antiserum directed against epitope tags inSmads. To determine expression levels of receptors, aliquots of celllysates were immunoprecipitated with antisera against type I receptors.Expression of Smads was determined by Western blotting on aliquots ofcell lysates.

Transcriptional Response Assay.

Mv1Lu wild-type and R mutant cells were transiently transfected withp3TPLux using DEAE-dextran transfection method, as described before(Nakao et al., 1997). HaCat cells were transiently transfected with p21reporter plasmid (Datto et al., J. Biol. Chem. 270: 28623-28628, 1995)using Transfectam reagent from Promega (Madison, Wis.). In eachexperiment, equal total amounts of DNA were transfected. In otherexperiments, Mv1Lu cells were transfected with p3TPLux and anti-senseexpression constructs using Lipofectin; after incubation overnight inOptimum medium, the medium was changed to serum-containing medium. Afteranother 24 h or incubation the cells were stimulated with TGF-β1 andluciferase activity was measured. Luciferase activity was measured aspreviously described (Nakao et al., 1997). The values were normalizedfor transfection efficiency using the β-gal reporter gene undertranscriptional control of CMV promoter (pCMV5 vector). Results shownare representative of at least three or four independent experiments.

Transient transfections of COS cells were performed using theDEAE-dextran protocol. Stable transfection of Mv1Lu cells with pMEP4expression vector (Invitrogen) was done using calcium phosphateprecipitation method, as previously described (Souchelnytskyi et al.,1997); selection was performed with 420 units/ml hygromycin. Inductionwith zinc chloride was done with 100 μM zinc chloride for 20 h, unlessindicated otherwise. Metabolic labeling of cells, [³²P]orthophosphatelabeling, and immunoprecipitation, and SDS-PAGE were performed asearlier described (Souchelnyskyi et al., 1997 or Nakao et al., 1997).

Xenopus Embryo Culture and Manipulation.

Xenopus eggs were obtained and embryos microinjected and cultured asdescribed (Moon and Christian, Technique 1: 76-89, 1989). For animal capassays, 200 pg of RNA encoding activin-β or a bone morphogeneticprotein-Vg1 chimera (Dale et al., EMBO J. 12: 4471-4480, 1993) wasinjected either alone, or together with 400 pg of Smad7 RNA. ForSmad7/Smad8 experiments, RNA was synthesized by in vitro transcription(Moon and Christian, 1989) of pCS2+Smad8 and pCS2+Smad7 (Nakayama etal., Development 125:857-867, 1998). The latter construct was generatedby subcloning the coding region of F-Smad7 into pCS2+ (Turner andWeintraub, Genes Dev. 8:1434-1447, 1994). Embryonic stages are accordingto Nieuwkoop and Faber (Normal table of Xenopus laevis, GarlandPublishing, Amsterdam, North Holland, 1967). Immunostaining of wholeembryos (12/101 antibody was obtained from the Developmental StudiesHybridoma Bank under contract N01-HD-2-3144 from the NICHD, Tor 70 wasobtained from R. Harland), whole mount in situ hybridization, RT-PCRanalysis of RNA extracted from cultured animal caps, and quantitation ofbrachyury expression relative to that of EF-la were performed asdescribed (Lagna et al., Nature 383: 832-836, 1996; Cui et al., Dev.Biol. 180: 22-34, 1996; Moon and Christian, 1989).

Cell Lines.

COS cells and Mv1Lu mink lung epithelial cells and HaCat cells (humankeratinocytes) were obtained from American Type Culture Collection.Cells were cultured in Dulbecco's modified Eagle's medium (LifeTechnologies, Inc., Gaithersburg, Md.) supplemented with 10% fetalbovine serum (FBS) and antibiotics (100 units of penicillin and 50 μg ofstreptomycin per ml). All the 10 different human lung carcinomas used[small cell lung carcinoma (SCLC), U-1285, U-1690, H-69 and H-92;non-SCLC, U-1752; squamous cell carcinoma (SQC); U-1810; large-cellcarcinoma (LCC), H-157, H-661; adenocarcinoma (ADC) H-23 and H-125(Histological typing of lung tumors, according to WHO, Geneva)] weregrown in RPMI 1640 medium with 10% FBS and antibiotics. (Heldin et al.,Br. J. Cancer, 68: 708-711, 1993).

RNA Isolation and Northern Blot Analysis.

Cells were kept in 0.5% FBS 12-24 h prior to stimulation with variousfactors followed by RNA extraction. Lung carcinoma cells used for RNAextraction were grown in the presence of 10% FCS. Isolation of total RNAand Northern blotting was performed essentially as described (Afrakhteet al., Int. J. Cancer, 68:802-809, 1996). The RNA extracted from thelung carcinomas cell lines was poly(A)⁺-enriched. Hybridizations wereperformed using QuickHyb buffer from Stratagene. The intactness andtotal amount of RNA (12 μg or total RNA or 5 μg of poly(A)⁺ RNA perlane) were checked by staining the gel with ethidium bromide and byhybridization with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)probe. The cDNA probes used in the hybridizations were a 1.8 kb Eco/XhoI human Smad7 fragment, a 2.0 kb EcoRI fragment of the human Smad6, a 3kb Eco RI fragment of the human PAI-1, and a 1.5 kb mouse JunB fragment.For the GAPDH hybridizations the entire human GAPDH cDNA plasmid waslabeled.

Antibodies

Antibodies against Smad2, termed DQQ, have been previously described(Nakao et al., 1997). Anti-Flag antibody was purchased from Kodak (NewHaven). Specific antisera for Smad6, termed ESP and SRQ were raisedagainst ESPPPPYSRLSPRDEYKPLD (SEQ ID NO:11) and SRQFITSCPCWLEILNPR (SEQID NO:12), respectively. Specific antisera raised against Smad7, termedKER and KAV, were raised against the synthetic peptidesKERQLELLLQAVESRGGTRTA (SEQ ID NO:13) and KAVRGAKGHHHPHPP (SEQ ID NO:14),respectively. The peptides were coupled to keyhole limpet haEmocyanin(Calbiochem-Behring) with glutaraldehyde, mixed with Freund's adjuvant,and used to immunize rabbits according to standard procedures.

Growth Inhibition Assay

Mv1Lu cells were seeded at 1×10⁴ cells/well in 24-well plates. Prior toaddition of TGF-β1 the Mv1Lu cells were simultaneously treated (or not)with 100 μM zinc chloride and indicated concentrations of TGF-β1 for 20h to induce Smad7 expression. Before harvesting, the cells were pulsedwith 0.2 μCi of [³H] thymidine (6.7 Ci/mmol, Amersham, UK) for 2h. Thecells were fixed with ice-cold 5% trichloroacetic acid (TCA) for morethan 20 min, washed twice with 5% TCA and once with water.Solubilization of the cells was done with 400 μl of 0.1 M NaOH for 20min at room temperature. The ³H-radioactivity incorporated into DNA wasdetermined by liquid scintillation counting.

Cells were grown in LAB TEK chambers (Nunc, Naperville, Ill.), andincubated with DMEM containing 0.3% FCS in the absence or presence ofTGF-β1 for 2 h. The slides were washed once with phosphate bufferedsaline (PBS), fixed for 10 min with 4% paraformaldehyde, followed bythree washes with PBS, subsequently permeabilized with 0.1% Triton X-100in PBS for 5 min, and again washed three times with PBS. Slides wereblocked by 10% goat serum for 1 h in room temperature, then incubatedwith 10% goat serum with anti-FLAG antibody (20 μg/ml) for 15 h at 4° C.The slides were subsequently washed 3 times, incubated withTRITC-conjugated goat anti-mouse IgG antibody (diluted 1:40) and washedagain 4 times. Nuclei were stained with DAPI (1 μg/ml) for 10 min inroom temperature followed by three washes. In order to visualize thefluorescence, a Zeiss microscope, or laser confocal microscope was used.For cell counting a square lattice mounted in one of the eyepieces wasused. In the COS cell experiments 200 cells were counted in 5 differentfield and the nuclear localization was checked by DAPI staining.

Example 1

Cloning and Characterization of Smad7

A database search for mammalian sequences related to Smads revealed theexistence of two mouse expression sequence tags (ESTs), i.e. AA022262and AA061644, with sequence similarity to N- and C-terminal Smaddomains, respectively. Polymerase chain reaction (PCR) using kidney cDNAas a template with a PCR primer pair derived from both ESTs resulted ina specific product. A cDNA encoding the complete mouse protein was madeby fusing mouse EST cDNA with a cDNA isolated from a mouse placentalibrary, and was termed mouse Smad7 (mSmad7). Human Smad7 cDNA (hSmad7)was isolated from a human brain cDNA library. The cDNA sequences predictthat mSmad7 and hSmad7 have 426 amino acid residues with 98% identity(FIG. 1a).

All differences between hSmad7 and mSmad7 are found in the N-terminaldomain. The borders of the Mad homology 2 (MH2) domain are indicated byarrows. The nucleotide sequences are deposited in European MolecularBiology Laboratory/GenBank data library (mouse and human Smad7 accessionnumbers are AF0152260 and AF015261, respectively). Smad7 is most relatedto Smad6 (Imamura et al., Nature, 389:622-626, 1997) with 36% and 56%sequence identities in the N-terminal domain and the C-terminal MH2domain, respectively. The Smad7 N-terminal domain, which contains aglycine/glutamic acid residue-rich region, shows only weak similarity(approximately 15%) to the MH1 domains found in Smad1 through Smad5.

RNA blot analysis of various tissues with a probe from the region codingfor the N-terminal domain of Smad7 revealed one major transcript ofapproximately 4.4 kb (FIG. 1b). Among the tissues analyzed, the highestexpression of Smad7 was found in the lung.

Example 2

Smad7 Modulates TGF-β Superfamily Signal Transduction

To investigate whether Smad7 modulates the responsiveness to TGF-β, theTGF-β-inducible luciferase p3TPLux reporter construct, which containsthe TGF-β-inducible PAI-1 promoter, was transfected into MV1Lu minkepithelial cells in the absence or presence of Smad7 cDNA. Smad7 wasfound to exert a dose-dependent inhibition TGF-β-induce luciferaseactivity (FIG. 2a). Moreover, the induction of p3TPLux luciferase by aconstitutively active variant of TβR-I, when transfected in R-mutantcells, was also inhibited by cotransfection with Smad7, as was theresponse by a constitutively active variant of the structurally relatedtype I receptor for activin (ActR-IB) (FIG. 2B). Transfection of Smad2did not affect TGF-β1-induced p3TPLux luciferase response in Mv1Lu cells(FIG. 2a). This inhibitory effect was specific as Smad7 did not inhibitthe phorbol 12-myristate 13-acetate (PMA)/epiderrnal growthfactor-induced p3TPLux luciferase response. In addition, theforskolin-mediated transcriptional induction using acAMP-responsive-element-containing reporter construct was not affectedby Smad7. These results indicate that Smad7 is a potent negativeregulator of both TβR-I- and ActR-IB-induced p3TPLux response.

To examine the effect of Smad7 expression on TGF-β-mediated growthinhibition, a luciferase transcriptional reporter construct containingthe p21 CDK inhibitor promoter (p21Lux) that is induced by TGF-β inhuman keratinocytes (HaCat) (Imamura, 1997) was used. Smad7 was found toantagonize TGF-β1-mediated p21 Lux response in HaCat cellsdose-dependently (FIG. 2c). Increased expression of Smad2 bytransfection slightly stimulated this response, whereas Smad1transfection had no effect (FIG. 2c). Smad7 and N-terminally Flag-taggedSmad7 gave essentially the same antagonistic results on TGF-β signaling,suggesting that N-terminal tagging does not interfere with functionalproperties of Smad7. Thus, Smad7 inhibits TGF-β-induced pathways leadingto extracellular matrix production as well as growth inhibition.

To determine whether Smad7 can inhibit transduction ofTGF-β/activin-like signals in vivo, patterning defects caused byoverexpression of Smad7 in Xenopus embryos were analyzed. When theendogenous activin signaling pathway is inactivated in early Xenopusembryos, by introduction of dominant negative forms of either an activinreceptor (Hemmati-Brivanlou et al., Nature 359: 606-614, 1992; Chang etal., Development 124: 827-837, 1997) or of Smad4 (Lagna et al., Nature383: 832-836, 1996), mesoderm fails to form. Similarly, microinjectionof RNA encoding Smad7 into both blastomeres of two-cell embryosinhibited mesoderm formation (FIGS. 2d-g). Five hundred picograms ofsynthetic RNA encoding either Smad7 or myc-epitope tag (MT) was injectedinto each blastomere of two-cell embryos as shown schematically (FIG.2d) and embryos were cultured to the tailbud stage. Embryos injectedwith MT RNA developed normally (top embryo in each panel) whereasembryos made to misexpress Smad7 (bottom embryo in each panel) failed toform head or tail structures (FIG. 2e) and showed a complete or partialloss of muscle (FIG. 2f) and notochord (FIG. 2g). Arrows indicateimmunoreactive muscle (FIG. 2f) and notochord (FIG. 2g) in MT-injectedembryos. Specifically, head and tail structures were absent or severelydeficient in 80% of injected embryos (n=168) (FIG. 2e) as weremesodermal derivatives such as muscle (n=60) (FIG. 2f) and notochord(n=48) (FIG. 2g).

To further test the ability of Smad7 to block mesoderm formation invivo, embryos were analyzed for expression of brachyury, a gene that isexpressed throughout the presumptive mesoderm during gastrulation (Smithet al., Cell 67:79-87,1991) (FIG. 2h). Five hundred picograms ofsynthetic RNA encoding MT (FIG. 2h) or Smad7 (FIG. 2i) was injected intoone blastomere of two-cell embryos near the equator and expression ofbrachyury analyzed by whole mount in situ hybridization. MT-injectedembryos show the typical ring of brachyury expression (FIG. 2h) whilebrachyury transcripts are not detected on one side of Smad7-injectedembryos (FIG. 2i, arrow). As shown in FIG. 2i, injection of Smad7 RNAinto the equatorial region of one blastomere of two-cell embryosprevented brachyury expression on one side of the embryo.

Two members of the TGF-β family, activin and VG1, are candidates forbeing endogenous mesoderm-inducing molecules (Kessler et al., Science266: 596-604, 1994). A Xenopus animal cap assay (Kessler, 1994) wasusedto directly test the ability of Smad7 to block signaling downstreamof these ligands. While both activin and Vg1 induced expression ofbrachyury in ectodermal explants (animal caps), coexpression of Smad7inhibited brachyury induction of 60%, thereby demonstrating that Smad7can block activin and Vg 1-mediated mesoderm induction.

To obtain insight into the mechanism by which Smad7 exerts its negativerole in signaling by TGF-β family members, it was tested whether Smad7,like Smad2 and Smad3 (Zhang et al., Nature 383:168-172, 1996;Macías-Silva et al., Cell 87:1215-1224, 1996; Nakao et al., 1997), canassociate with the TGF-β receptor complex. COS cells transfected withN-terminally Flag-tagged Smad7 in combination with TβR-II (wild-type,WT, or kinase-deficient mutant, KD) and TβR-I (wild-type or kinasedeficient mutant) were affinity labeled with ¹²⁵I-TGF-β1, and celllysates were subjected to immunoprecipitation with Flag antiserumagainst the Flag-epitope in Smad7. Immunoprecipitates were analyzed bySDS-PAGE and FujiX Bio-Imager. Expression of receptors and F-Smad7 aftertransfection was determined by immunoprecipitation with VPN antibodyagainst TβR-I or DRL antibody against TβR-II on aliquots of celllysates, and by immunoprecipitation with Flag antibody on lysates [³⁵S]methionine/cysteine labeled transfected cells. It was found that Smad7interacted very efficiently with the TGF-β receptor complex (FIG. 3).Smad7 interacted with wild-type TβR-I as well as kinase inactive TβR-Iin complex with wild-type TβR-II. In contrast, Smad2 and Smad3 interactstably only with complex of kinase-deficient TβR-I and wild-type TβR-II(Macías-Silva et al., 1996; Nakao et al., 1997). No interaction wasobserved between Smad7 and a heteromeric complex of kinase-deficientTβR-II and TβR-I, or with TβR-II alone (FIG. 3). Thus,transphosphorylation of TβR-I by TβR-II kinase is required forassociation of Smad7 with TβR-I.

Example 3

Phosphorylation of Smad7

Phosphorylation of Smad2 and Smad3, but not Smad4, is induced afterTGF-β stimulation (Eppert et al., Cell 86: 543-552, 1996; Zhang et al.,1996; Macías-Silva et al., 1996; Nakao et al., 1997; Lagna et al., 1996.It was investigated whether Smad7 is phosphorylated upon associationwith TGF-β receptors. COS cells were transfected with Smad7 (or forcomparison with Smad2) in the absence or presence of receptors, labeledwith [³²P]orthophosphate, and treated with TGF-β1. Cell lysates weresubjected to immunoprecipitation with antisera against Smads or epitopetags, and the level of Smad phosphorylation was determined. Expressionof type I receptors and Smads was analyzed by immunoprecipitation onaliquots of cell lysates, which had been labeled with[³⁵S]-methionine/cysteine. The ³²P- or ³⁵S-radioactivity associated withSmad2in (FIG. 4b) or Smad3 in (FIG. 4d) were quantitated by using aFujiX Bio-Imager and the 32P/³⁵ S-ratio calculated (FIGS. 4c and e,respectively). Representative results of three independent experimentsare shown. As expected, Smad2 phosphorylation was increased uponcoexpression with receptors, and addition of TGF-β1 led to a furtherincrease of Smad2 phosphorylation. However, no (or only very weak)phosphorylation of Smad7 was observed in cells transfected withconstitutively active forms of TβR-I and ActR-IB or in cells transfectedwith wild-type TβR-I and TβR-II after stimulation with ligand (FIG. 4a).Thus, in spite of its direct association with TβR-I, Smad7 is not adirect substrate for the TβR-I kinase. Notably, Smad7 lacks theconserved SS(M/V)S C-tail sequence motif (FIG. 1a), which in the case ofSmad2 (Macías-Silva et al., 1996) and Smad1 (Kretzschmar et al., GenesDev. 11: 984-995, 1997) are phosphorylated by type I receptor kinase.

Example 4

Smad7 Reduces Phosphorylation of Smad Proteins

Smad7 may exert its negative role in TGF-β signaling by interfering withactivation of other Smads. Therefore it was examined whether Smad7affects the phosphorylation of Smad2 and Smad3 using[³²P]orthophosphate-labeled COS cells transfected with TGF-β receptorsand Smads. COS cells were transfected with F-Smad7 or Smad2 (FIG. 4a),Smad2 alone or together with Smad7 (FIG. 4b), and N-terminallyMyc-tagged Smad3 (M-Smad3) alone or together with F-Smad7 (FIG. 4d) inthe absence or presence of wild-type or constitutive active (c.a.)receptors, whereafter cells were labeled with [³²P]orthophosphate.C-terminally HA-tagged type I receptors were used. Interestingly, Smad7abolished the TGF-β1-induced phosphorylation of Smad2 (FIGS. 4b,c) andSmad3 in dose-dependent manner (FIGS. 4d,e). A Smad7 level twice as highas Smad3 appeared sufficient to cause a significant decrease inTGF-β-induced receptor-mediated Smad3 phosphorylation. As previouslyreported (Macías-Silva et al., 1996; Nakao et al., 1996), it was foundthat Smad2 and Smad3 become phosphorylated when overexpressed in COScells. For Smad2 this receptor-independent phosphorylation does notoccur at the SSMS motif in the C-terminal tail (Macías-Silva et al.,1996), and may possibly occur via non-receptor kinases. Smad7 appearsnot to block this receptor-independent phosphorylation. Since activationof Smad2 and Smad3 is essential for optimal TGF-β signaling (Zhang etal., 1996; Macías-Silva et al., 1996; Nakao et al., 1997; Lagna et al.,1996; Kretzschmar et al., 1997), the inhibition of their phosphorylationprovides a mechanistic explanation for the antagonistic effect of Smad7.The association of Smad7 with TβR-I suggests that it may compete withSmad2 and Smad3 for receptor binding. In accordance with this notion,co-transfection of Smad2 or Smad3 with Smad7 reduced the antagonisticeffect of Smad7 in a TGF-β1-induced p3TPLux assay. In addition, it wasdetermined that Smad7 not only inhibits TGF-β and activin signaling, butalso blocks BMP signaling. Using methodology described herein, it wasdetermined that Smad7 associated with BMPR-Is and inhibitedphosphorylation of SmadI in cultured mammalian cells. In addition,injection of Smad7 RNA into ventral cells of Xenopus embryos phenocopiesthe effect of blocking the BMP signaling pathway, and led to formationof an incomplete secondary dorsal axis.

Example 5

Regulation of Smad7 Expression by TGF-β

Regulation of Smad7 expression by signaling molecules may be used toeffectively modulate TGF-β responses. Therefore, it was investigatedwhether the expression of Smad7, and for comparison Smad2, Smad3 andSmad4, were regulated by TGF-β1. Northern blots with 20 μg/lane totalRNA from TGF-β1 stimulated Mv1Lu cells, SW1736 human anaplastic thyroidcarcinoma cells and HaCat cells probed with Smad7 revealed that Smad7mRNA was rapidly induced in response to 10 ng/ml TGF-β1 stimulation(FIGS. 5a,b). The amount of RNA loaded was checked by hybridization offilters to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAprobe. In Mv1Lu cells, Smad7 mRNA was induced 4-fold after 30 min ofTGF-β stimulation. Expression of Smad2, Smad3 and Smad4 upon TGF-β1stimulation remained unchanged (FIG. 5a). Induction of Smad7 mRNA byTGF-β1 was observed in the presence of 20 μg/ml cycloheximide (CHX),indicating that de novo protein synthesis is not required forTGF-β1-mediated induction of Smad7. In fact, Smad7 mRNA was superinducedin the presence of TGF-β1 and 20 μg/ml CHX added 30 min. prior to TGF-β1(FIG. 5b). This is likely caused by increase in mRNA stability or lossof transcriptional repressors by CHX. Treatment with CHX alone onlyslightly increased the basal Smad7 mRNA level. In contrast, 10 nM PMA,which like TGF-β1 stimulates PAI-1 expression and inhibits the growth ofMv1Lu cells, did not induce Smad7 expression (FIG. 5c). In Mv1Lu cellslacking functional TβR-I (R mutant), TGF-β1 had no effect on Smad7 mRNAexpression. Taken together these data indicate that Smad7 is animmediate-early response gene for TGF-β1, and activated complexes ofSmad2, Smad3 and Smad4 may directly and/or in combination with DNAbinding cofactors act on the Smad7 promoter. The intracellularantagonist Smad7 may thus act in a negative feedback loop to regulatethe intensity or duration of the TGF-β signal.

Example 6

Smad7 Interacts with BMP Receptor Complexes and Inhibits BMP-MediatedSignaling.

To determine if Smad7 associates with the BMP receptor complex, COScells were transfected with the BMP receptors BMPR-IA, BMPR-IB, orActR-I (wild-type or kinase-deficient mutant) tagged at C-terminus withHA epitope, and BMPR-II (wild-type or kinase-deficient mutant), in theabsence or presence of N-terminally Flag-tagged Smad7 (F-Smad7). Thereceptors were covalently affinity labeled with ¹²⁵I-BMP-7, and celllysates were subjected to immunoprecipitation with Flag antibody toimmunoprecipitate F-Smad7. Immunoprecipitates were analyzed by SDS-PAGEand FujiX Bio-Imager. Expression of receptors and F-Smad7 (not shown)after transfection was determined by immunoprecipitation withHA-antisera on aliquots of cell lysates, and by immunoprecipitation withFlag antibody on lysates from [³⁵S]methionine/cysteine labeledtransfected cells. As shown in FIGS. 6A-F, TGF superfamily receptorscoimmunoprecipitated with F-Smad7. Therefore, Smad7 associated withBMPR-IA and BMPR-IB, and less efficiently with ActR-I receptors. Asshown in FIG. 6B, no interaction was observed between Smad7 andheteromeric complexes of a kinase-deficient type II receptor and awild-type or a kinase-deficient type I receptor. Thus Smad7 onlyinteracts with type I receptors which are phosphorylated by the type IIreceptor (i.e. activated). Among the tested type I receptors, Smad7preferentially interacts with BMPR-IA, BMPR-IB, TβR-I and likelyActR-IB, and less efficiently with ActR-I. In addition, the effect ofSmad7 on BMPR-IB- and ActR-I-mediated phosphorylation of Smad1 or Smad5was examined (FIG. 6C-F). FIG. C shows that Smad 7 inhibits BMPR-IB- andActR-I-mediated phosphorylation of Smad1. COS cells were transfectedwith F-Smad1 alone or together with F-Smad7 in the presence of type Ireceptors (BMPR-IB or ActR-I) and BMPR-II in the absence or presence ofBMP-7, whereafter cells were labeled with [³²P]orthophosphate andimmunoprecipitated with Flag antisera. Expression of F-Smad1 and F-Smad7was determined by immunoblotting with Flag on aliquots of cell lysates.FIG. 6D shows that the ³²P-radioactivity associated with Smad1 in FIG.6C as quantified by using a FujiX Bio-Imager and plotted. FIG. 6E showsthat Smad7 inhibits BMPR-IB phosphorylation of Smad5. COS cells weretransfected with F-Smad5 alone or together with F-Smad7, in the presenceof BMPR-IB and BMPR-II; cells were incubated in the absence or presenceof BMP-7, whereafter they were labeled with [³²P]orthophosphate andsubjected to immujnoprecipitation with Flag antisera. FIG. 6F shows the³²P-radioactivity associated with Smad5 in FIG. 6E as quantified using aFujiX Bio-Imager and plotted. COS cells were transfected with cDNAsencoding BMP receptors and Smad1 or Smad5 in the absence or presence ofSmad7. The phosphorylation level of Smad1 or Smad5 was examined by[³²1′]orthophosphate labeling and immunoprecipitation from cell lysatesusing Smad antisera. Smad7 potently inhibited BMPR-IB-mediatedphosphorylation of Smad1 and Smad5 in a dose dependent manner (FIGS.6C-F). In addition, Smad7 inhibited ActR-I-mediated phosphorylation ofSmad1 (FIGS. 6C, D). The specificity of the interaction based on thephosphorylation of the type I receptor by type II receptor kinasestrongly suggests physiological importance of the interaction.Phosphorylation may induce a conformational change that is required forSmad7-type I receptor interaction.

To determine if Smad7 inhibits BMP-mediated phosphorylation of pathwayspecific Smads, COS cells were transfected with F-Smad1 or F-Smad5,alone or together with F-Smad7 in the absence or presence ofconstitutive active (c.a.) BMPR-IB (FIG. 7A), or alone or together withF-Smad7, type I receptors and BMPR-II in the absence or presence ofBMP-7 (FIG. 7C), whereafter cells were labeled with[³²P]-orthophosphate. The amount of transfected Smad7 is indicated inFIG. 7C (0.1 or 1.0 microgram). Cell lysates were subjected toimmunoprecipitation with antisera against Smads, and the level ofphosphorylation determined. Expression of Smads and BMPR-IB was analyzedby immunoprecipitation with antisera directed towards epitope tags onaliquots of cell lysates, which had been labeled with[³⁵S]methionine/cysteine. In FIG. 7B, the ³²P or ³⁵S-radioactivityassociated with SmadI were quantitated by using FujiX Bio-Imager and the³²P/³²S-ratio calculated. FIG. 7 shows that Smad7 potently inhibitedphosphorylation of by BMPR-IB-mediated Smad1 or Smad5 phosphorylation ina dose-dependent manner. Consistent with the differential receptorbinding, BMPR-IB-mediated Smad phosphorylation was more efficientlyinhibited by Smad7 than was ActR-I-mediated Smad phosphorylation.

Example 7

Analysis of Smad7 N-terminal and C-terminal Domain Function

To determine if the C-terminal domain of Smad7 associates with the TGF-βreceptor complex, COS cells were transfected with wild-type N-terminallytagged mouse Smad7 (F-mSmad7) or a N-terminally Flag tagged mouse Smad7C-terminal domain (amino acids 204-426 of mSmad7) in combination withwildtype TGF-β receptors. The receptors were covalently affinity labeledwith ¹²⁵I-TGF-β1. Immunoprecipitates were analyzed by SDS-PAGE and FujiXBioImager. Expression of receptors and Smad7 after transfection wasdetermined by immunoprecipitation with VPN antisera against TβR-I onaliquots of cell lysates, and by immunoprecipitation with Flag antibodyon cell lysates from ³⁵S methionine/cysteine labeled transfected cells.As shown in FIG. 8A, the TGF-β receptor complex coprecipitates withSmad7 C-terminal domain. Therefore, the Smad7 C-terminal domainassociates with TGF-β receptors. Next, the C-terminal domain was testedin an assay of TGF-β-induced p3TPLux response, as described above. Asshown in FIG. 8B, the C-terminal domain inhibits, albeit lessefficiently as wild-type Smad7, the TGF-B-induced p3TPLux response. FIG.8B, panel 2 shows that transfection of Smad7C blocks TGF-β-inducedp3TPLux transcriptional response, but less efficiently than will-typeSmad7.

The inhibitory effect of N- and C-terminal Smad7 domains and Smad8 onTGFβ superfamily receptor responses was tested. Smad8 and Smad7 deletionconstructs were tested for effects on TβR-I-mediated phosphorylation ofSmad2 or Smad3. COS cells were transfected with TβR-I and TβR-II andindicated Smads. Phosphorylation of Smads was determined by a ³²Porthophosphate labeling assay. FIG. 8C shows that Smad8 and N- andC-terminal (MH2) domains of Smad7 each inhibited TβR-I-mediatedphosphorylation of Smad2 or Smad3. The effect of the C-terminal domainwas achieved at lower expression levels than the N-terminal domain.Consistent with the ability of the C-domain of Smad7 to inhibitTβR-I-mediated phosphorylation of Smad2, this domain was sufficient toinhibit TGF-β-induced transcriptional activation, although it was lessefficient at doing so than was wild-type Smad7 (FIG. 8B, panel 2).Deletion of the last 19 amino acids residues completely abrogated theability of the Smad7 C-domain to inhibit TGF-β-induced transcriptionalactivation of the 3TP promoter.

FIG. 8D shows the inhibitory effect of Smad7 N- and C-terminal domainsand Smad8 (as compared to Smad7 wild-type) on TGF-β receptor mediatedp3TPLux transcriptional response. Mv1Lu cells were transfected withp3TP-Lux reporter construct with or without different quantities ofinhibitory Smads. The induction of luciferase expression upon cellincubation with TGF-β was measured. Consistent with their differentialphosphorylation inhibitory effects, Smad8 and the Smad7 domainsinhibited the TGF-β mediated transcriptional response of the reportergene.

FIG. 8E shows that Smad7C inhibits TβR-I-mediated phosphorylation ofSmad2, but less efficiently than wild-type Smad7. In the absence ofinhibitory Smad, TGF-β1 stimulated a 1.6-fold increase in Smad2phosphorylation level, which was completely blocked by cotransfectionwith 1 μg wild-type Smad7, whereas in the presence of 1 μg Smad7C a1.2-fold increase in TGF-β-mediated Smad2 phosphorylation was observed.COS cells were transfected with F-Smad2 alone or together with F-Smad7Cor wild-type F-Smad7 in the presence of BMPR-IB and BMPR-II; cells werethen incubated in the absence or presence of BMP-7, whereafter they werelabeled with [³²P]orthophosphate and subjected to innmunoprecipitationwith Flag antisera. Expression of F-Smad2, F-Smad7 and F-Smad7C wasdetermined by immunoblotting with Flag on aliquots of cell lysates.

Example 8

Smad6 is Highly Conserved and Most Closely Related to Smad7

A database search for human and mouse sequences related to Smad7revealed the existence of an expressed sequence tag (EST; Genbankaccession number N95582) corresponding to a novel human Smad. Byscreening of a human placenta cDNA library we obtained a 3.2 kb cDNAclone. The entire coding region of this clone was sequenced using ABIPrism 310 Genetic Analyzer, and sequence analysis was performed withDNASTAR. Sequence analysis revealed 92% amino acid similarity to mouseSmad6 (Imamura et al., Nature, 389:622-626, 1997), and therefore thisgene was termed human Smad6 (hSmad6) (FIG. 9A). FIG. 9A shows sequencecomparison of human and mouse Smad6 and human Smad7. Identical residuesare boxed. The borders of the Mad-homology MH2 domain are indicated byarrows. The Genbank accession number for human Smad6 is AF043640. Thesequence identity between human and mouse Smad6 is lower compared tothat observed with other Smads, e.g., human and mouse Smad7 are 98%identical (Hayashi et al., 1997; Nakao et al., 1997). FIG. 9B showspairwise alignment relationship between hSmad1 through hSmad7 andhSmad9. Among the human Smads identified to date, hSmad6 is most closelyrelated to hSmad7 (41% overall sequence identity). Like the N-terminaldomain of Smad7, the N-terminal domain of Smad6 shows very weaksimilarity to MH1 domains from Smad1 through Smad5. Notably hSmad6 lacksthe conserved SS(MNV)S motif in its carboxy-terminal tail, which in thecase of pathway-restricted Smads is phosphorylated by appropriate type Ireceptor kinases. Recently Hata et al. reported a hSmad6 sequence(accession number AF035528) (Hata et al., Genes Dev., 12:186-197, 1998).Comparison of the two sequences reveals one nucleotide difference in thecoding region resulting in one amino acid difference (codon 21 inAF043640 is predicted to be an aspartic acid residue).

Example 9

Smad6 and Smad7 Expression in Lung Cancer Cell Lines

As previously reported, the distribution of Smad6 mRNA in various humantissues revealed that Smad6 was broadly expressed in various humantissues. One Smad6 transcript of approximately 3 kb was detected(Imamura, T., et al. 1997). Interestingly, the expression profiles ofSmad6 and Smad7 (Nakao et al., 1997) were very similar with the highestexpression in the lung. Therefore, a panel of 10 different lungcarcinoma cell lines (four SCLC and six non-SCLC) was examined for Smad6and Smad7 mRNA expression (FIG. 9C). The highest Smad6 mRNA expressionwas detected in the SCLC cell lines H-69 and H-82, and in the non-SCLCcell lines H-661 and H-23, whereas the SCLC cell line U-1690, H-69 andH-82, and the non-SCLC cell lines H-157 and H-125 expressed the highestlevels of Smad7 mRNA. Thus, the two genes are differently expressed, andno correlation was observed between Smad6 and Smad7 expression in thesecells.

Example 10

TGF-β Family Members Induce Smad6 and Smad7 mRNA in Mv1Lu and HaCatCells

Mink lung epithelial (Mv1Lu) and human keratinocyte (HaCat) cell linesare responsive to TGF-β1, activin and BMP-7 (Yashamita et al. J. CellBiol. 130:217-226,1995). Smad7 mRNA is rapidly included by TGF-β1(Nakao, A., 1997). Northern blot analysis of Smad6 and Smad7 expressionwas performed on RNA prepared from Mv1Lu and HaCat cells stimulated withTGF-β1 (10 ng/ml), activin (50 ng/ml) and BMP-7 (500 ng/ml). PhosphorImager quantitation revealed that (after 90 min of ligand stimulationand normalizing with GAPDH mRNA expression level) in Mv1Lu cells (FIG.10A Panel 1), TGF-β1, activin and BMP-7 induced Smad6 mRNA expression6-, 2- and 3-fold, respectively, and induced Smad7 mRNA expression 5-,2-, and 3-fold, respectively, and in HaCat cells (FIG. 10A Panel 2),TGF-β1, activin and BMP-7 induced Smad6 mRNA expression 4-, 2- and12-fold, respectively, and included Smad7 mRNA expression 6-, 3-, and6-fold, respectively. Both genes were rapidly induced by all threeligands (FIG. 10A). Smad6 and Smad7 were induced with similar kineticsby TGF-β and activin, with a major peak of expression after 90 min ofstimulation. A second peak of Smad6 and Smad7 mRNA expression wasobserved after 24 hr of stimulation Mv1Lu cells, in particular afterTGF-β1 stimulation (FIG. 10A). There was a difference in theBMP7-induced mRNA expression profiles for Smad6 and Smad7 in Mv1Lucells: whereas Smad7 mRNA expression peaked at 90 min and was reduced tolow levels thereafter, Smad6 mRNA remained high after 90 min ofstimulation (FIG. 10A). Treatment of cells with 0.5% FCS, prior toligand treatment, was found to decrease the basal level of inhibitorySmad expression, making the mRNA inductions observed with ligands morepronounced. The expression of Smad6 and Smad7 mRNA after stimulationwith ligands were similar in cultures of high and low cell density.

As shown in the Examples above, Smad7 not only inhibits TGF-β andactivin, but also BMP signaling. Smad7 associates with BMP type Ireceptors and inhibits BMPR-I-mediated signaling. Thus Smad7 may exert anegative feedback control on signaling by TGF-β1, activin as well asBMP-7.

Example 11

Smad6 is a Direct Target Gene for TGF-β1

Previously it was demonstrated that the induction of Smad7 mRNA byTGF-β1 was observed in the presence of cycloheximide (CHX), indicatingthat de novo protein synthesis is not required for this response.Induction by TGF-β1 of Smad6 mRNA was also seen after addition of CHX(FIG. 10B). CHX (20 μg/ml) was added 30 min before TGF-β1. The inductionof Smad6 mRNA was prolonged in the presence of both TGF-β1 and CHX whencompared to TGF-β1 alone (FIG. 10B), probably as a result of CHX-inducedloss of transcriptional repressors or increase in Smad6 mRNA stability.Based upon these results, Smad6 and Smad7 transcription are likely to bedirectly regulated by pathway-restricted and common mediator Smads.Therefore, the effect of TGF-β1 on MDA-MB-468 breast cancer cells thatlack Smad4 (Lagna et al., Nature, 383:832-836, 1996) was examined (FIG.10C). Smad6 mRNA expression was not affected by TGF-β1 in these cells.Smad7 expression was below detection level in the absence or presence ofTGF-β1, or possibly the Smad7 gene may be absent in these cells. Takentogether, the results indicate that Smad6 and Smad7 are immediate-earlyresponse genes for TGF-β family members.

Example 12

Transfection of Anti-sense Expression Construct Enhances CellularResponsiveness to TGF-β1

To investigate whether the expression level of inhibitory Smads modulatethe responsiveness to TGF-β, TGF-β1-induced transcriptional response wasmeasured in Mv1Lu cells transfected with an anti-sense Smad7 cDNAexpression construct. MV1Lu cells that are grown in the presence ofserum have relatively high constitutive Smad7 expression (FIG. 9C), andSmad7 is rapidly induced by TGF-β1 (FIG. 10A). Transfection ofanti-sense Smad7 expression construct (mSmad7r), but not empty vector oranti-sense Smad3 expression construct (hSmad3r), increasedTGF-β1-mediated p3TPLux transcriptional response in Mv1Lu cells. TGF-β1(10 ng/ml) was added 4 h prior to lysis of cells to measure luciferaseactivity. Data are presented as mean +/−SEM. Transfection of Mv1Lu cellswith 20 μg of anti-sense Smad7 expression plasmid increased the TGF-β1response, measured as activation of the PAI-1 promoter containingreporter, (p3TPlux) after a 4 h stimulation. The increased TGF-βresponse in the Smad7 anti-sense transfected cells was 2-4 fold comparedto control cells transfected with empty expression vector (FIG. 11); theresponse was dependent on the amount of transfected anti-senseexpression construct. Transfection with anti-sense Smad3 expressionconstruct showed no or very weak inhibitory response (FIG. 11). Theseresults indicate that the expression level of Smad7 determines thecellular responsiveness to TGF-β1.

Example 13

Effect of EGF and PMA on Smad6 and Smad7 mRNA Expression

To examine the possibility of cross-talk between different signalingpathways, the effects of forskolin (activator of cAMP), epidermal growthfactor (EGF) and phorbol ester PMA responses were determined in theabsence and presence of 10 ng/ml TGF-β1. Forskolin had no effect alone,and only slightly enhanced the TGF-β1-induced expression of Smad6 andSmad7 mRNA. EGF (10 ng/ml) by itself was capable of inducing Smad6 andSmad7 expression, and acted synergistically with TGF-β1 for theinduction of Smad7 mRNA (FIG. 12A). EGF was added 60 min. before TGF-β1.PMA (10⁻⁸ M) alone had no appreciable effect, but a strong synergisticresponse was observed when PMA was added together with TGF-β1 on theinduction of Smad6 and Smad7 expression (FIG. 12B). In this particularexperiment the TGF-β1-mediated induction of Smad6 and Smad7 mRNA wasvery low, but could be observed upon longer exposure to X-ray film,thereby making synergistic effect more apparent. PAI-1 gene expression,which was found to be more rapidly induced by TGF-β1 (peak at 90 min)than PMA (peak at 4 h), was included as a control to show effect of PMAand TGF-β1 on cells.

Example 14

Smad7 is More Potent than Smad8 In Inhibiting TGF-β-induced Responses

Xenopus Smad8 shares 96% amino acid identity with murine Smad7 withinthe C-domain (Nakayama et al, 1998). Given that this domain issufficient for receptor binding, and for inhibition of severalTGF-β-induced responses, one would predict that these proteins wouldbehave identically with regard to receptor binding and inhibition ofdownstream signaling. Consistent with this possibility, Smad8 interactedwith the activated TβR-I, BMPR-IA, BMPR-IB and ActR-I. In addition,Smad9 inhibited BMPRI-mediated phosphorylation of Smad1 and Smad5 in adose-dependent fashion that was indistinguishable from that of murineSmad7. FIG. 13A shows that Smad8 inhibits T βR-I-mediatedphosphorylation of Smad2 less efficiently than Smad7. Cells weretransfected with F-Smad2 alone or together with F-smad7 or F-Smad8 inthe presence of T βR-I and T βR-II; cells were then incubated in theabsence or presence of TGF-β. The level of F-Smad2 phosphorylation wasdetermined by [³²P]orthophosphate labeling and immunoprecipitation ofcell lysates with Flag antisera. Expression of F-Smads was analysed byWestern blotting with Flag antiserum on aliquots of cell lysates. FIG.13B shows that transfection of Smad7 in Mv1Lu cells blocks theTGF-β-induced p3TPLux response more effectively than Smad8. Thus, whileSmad7 efficiently inhibited TβR-I-mediated phosphorylation of Smad2,Smad8 was less effective at doing so (FIG. 13A). Interestingly, thiscorrelated with the more pronounced effect of Smad7 versus Smad8 ininhibiting TGF-β-induced transcriptional activation of the p3TPLuxreporter gene (FIG. 13B). Of note, Smad7 is more effective than is Smad8at inhibiting activin signaling in Xenopus mesoderm induction assay(Nakayama et al., 1998).

Example 15

Partial Secondary Axis Formation by Smad7 and Smad8

To determine whether Smad7 can inhibit transduction of BMP signals invivo,patterning defects caused by overexpression of Smad7 in Xenopusembryos were analyzed. FIG. 14A shows photomicrographs of controltadpole and FIG. 14B shows sibling tadpole made to misexpress Smad7 inventral cells. Note induction of partial secondary dorsal axis (arrow).FIG. 14C and FIG. 14D (control) depict Smad7-RNA injected siblingshowing immunoreactive muscle in primary (arrow) and secondary(arrowhead) axes. FIG. 14E shows eye defects and spina bifida in anembryo made to overexpress Smad8 in dorsal cells. FIG. 14A shows fusionof eyes in an embryo made to overexpress Smad7 in dorsal cells.Overexpression of known BMP antagonists, such as dominant negative BMPreceptors or ligands, on the ventral side of Xenopus embryos can inducethe formation of a partial secondary axis (reviewed inGraff Cell89:171-174, 1997). Injection of 200 or 400 pg of RNA encoding Smad7 nearthe ventral midline of four-cell embryos led to formation of a secondarydorsal axis in 90% (n=100) or 96% (n=79) of embryos, respectively (FIG.14B). Secondary axis formation was observed in 94% (n=98) of embryosinjected with 200 pg of RNA encoding that isolated C-domain of Smad7.Axes induced by either Smad7 or the C-domain of Smad7 containedimmunoreactive muscle (FIG. 14D) but lacked notochord. In the sameseries of experiments, injection of 200 or 400 pg of RNA encodingXenopus Smad8 near the ventral midline induced the formation of asecondary axis in 87 (n=92) or 91% (n=101) of injected embryos,respectively. One notable difference between the axes induced by the twogene products is that the secondary axes induced by Smad8 included acyclopic or fused eye in 10% (200 pg) or 41% (400 pg) of cases whereaseye formation was rarely observed (3% of embryos) followingmisexpression of Smad7, and then only when a high dose (400 pg) of RNAwas injected.

Example 16

Ectopic Expression of Smad7 in Dorsal Cells Induces a Subset of thePatterning Defects Caused by Overexpression of Smad8

Ectopic expression of Smad8 within dorsal cells of Xenopus embryosproduces a range of phenotypic defects which cannot be attributed toblockade of BMP signaling. These include failure of the neural folds tofuse (spina bifida) as well as a range of eye defects (Nakayama et al.,1998). To directly compare patterning defects cause by misexpression ofSmad7 and Smad8 within dorsal cells, 200 pg of RNA encoding eitherprotein was injected near the dorsal midline of four-cell embryos. While20% of Smad8-injected embryos developed spina bifida, and 40% showed adecrease in eye pigmentation (n=50), 97% of Smad7-injected embryosdeveloped completely normally (n=89). When a higher dose (400 pg) of RNAencoding either Smad8 or Smad7 was injected, 40% of Smad8-injectedembryos developed with spina bifida (FIG. 14E, n=124) while only 7% ofembryos injected with Smad7 RNA (n=140), and 3% of embryos injected witha control RNA (encoding a myc epitope tag, n=70) displayed this defect.In contrast, 43% of Smad7-injected embryos developed with variable eyedefects ranging from a slight to moderate decrease in eye pigment tofusion of the eyes (FIG. 14F). Forty-seven per cent of Smad8-injectedsiblings developed with comparable eye defects (Nakayama et al., 1998).The percentage of Smad8-injected embryos displaying spina bifida, eyeinduction or eye defects was somewhat lower in these experiments thanthat previously reported (Nakayama et al., 1998), possibly due toinherent differences between embryos used for the two sets ofexperiments. Together, these results suggest that Smad7 and Smad8 caninteract with common signaling pathways in vivo although Smad8 maytarget additional, as of yet unidentified, pathways. Therefore,overexpression of Smad8 in Xenopus embryos produces patterning defectsthat are not observed following overexpression of Smad7, suggesting thatSmad7 and Smad8 may preferentially target distinct signaling pathways.While it cannot be ruled out that the possibility that these differencesare due to species specificity, e.g., the possibility that mammalianSmad7 is more efficiently translated, or interacts more efficiently withreceptors in mammalian cells than does Xenopus Smad8, the results do notsupport this interpretation. Specifically, Smad7 is a more potentantagonist of TGF-β/activin signaling in both mammalian and Xenopussystems than is Smad8.

Example 17

Smad7 but not Smad6 inhibits TGF-β1-induced Growth Inhibition.

In order to investigate the effect of inhibitory Smads onTGF-β1-mediated growth inhibition, mouse Flag-tagged Smad6 (F-Smad6) andF-Smad7 were stably transfected into Mv1Lu cells; Smad6 and Smad7 wereplaced under transcriptional control of the human induciblemetallothionien IIA promoter using the pMEP4 expression vector. Bothreported forms of Smad6 were tested; a mouse long Smad6 version (Smad6L;Imamura et al., Nature 398:622-626, 1997) as well as a human short Smad6version (Smad6S; Topper et al., PNAS 94:9314-9319, 1997). FIG. 15 showscharacterization of Smad expression level in stable transfected Mv1Lucell lines in the absence or presence of zinc chloride; wild-typeF-Smad7 (pMEP4-smad7; three independent clones, 7-3, 7-5S and 7-10), theN-terminal domain of Smad7 (pMEP-7N, clone 7N-5), Smad7 with deletion atC-tail (pMEP4-7NCΔ, clone 7NCΔ-7), the C-terminal domain of Smad7(pMEP4-7CΔ, clone 7CΔ-7) and the “short” variant of Smad6 (pMEP4-Smad6S,clone S6-2)and the “long” variant of Smad6 (pMEP4-Smad6L, clone L6-3)expression results are shown. Cells were metabolically labeled, and celllysates subjected to immunoprecipitation with Flag antibody.Immunoprecipitates were analyzed by SDS-PAGE. When the expression ofSmad6S, Smad6L and Smad7 was analyzed, we observed that the inhibitorySmads were already expressed in the absence of the inducer zincchloride. This is result of leakage of the metallothionin promoter.Pretreatment of cells with zinc chloride in each transfectant led toincrease in Smad6 and Smad7 expression (FIG. 15). Subsequently, wetested the response of the Smad7 and Smad7 (and empty pMEP4)transfectants for TGF-β1-induced growth inhibition in the absence orpresence of zinc chloride. Multiple independent Smad7-expressing cloneswere analyzed. We found that Smad7 prevented the TGF-β1-inducedgrowth-inhibitory effects in Mv1Lu (FIGS. 16A-C). In all Smad7 clonesTGF-β1-induced growth inhibition was blocked to an extent whichcorrelated with their different expression levels of Smad7.pMEP4-transfected Mv1Lu cells, in the absence or presence of zincchloride showed a similar dose response curve for TGF-β1 asnontransfected Mv lLu cells. Ectopic expression Smad6L or Smad6S did notaffect the TGF-β1-induced growth inhibition (FIGS. 16D,E). FIG. 16 showsthe effect of TGF-β1 on pMEP4 transfected cells in the absence orpresence of zinc chloride. The relative growth compared to control isplotted against the concentration of TGF-β1. Different lots of TGF-β1were used in experiments A-C versus D,E, explaining why there is adifference between the ED50's on pMEP4 transfected cells in theseexperiments. All data shown are means +/−SED. Thus Smad7 can antagonizeTGF-β1-induced growth inhibition, and appears to be more effective thanSmad6.

Example 18

Smad7 Inhibits TGF-β1-induced Transcriptional Responses.

Subsequently we examined the effect of ectopic Smad7 expression onTGF-β1-induced expression of the endogenous early response genes, Smad7,PAI-1 and JunB (FIG. 17). In FIG. 17 it is shown that ectopicallyexpressed Smad7 inhibits TGF-β1-mediated induction of JUNB (A), Smad7(B) and PAI-1 mRNA (E) expression. Northern blot analysis on RNA fromMv1Lu cells stably transfected with pMEP4-Smad7 without or withpretreatment with zinc chloride on exposure to TGF-β1. The endogenousmRNAs for junB, Smad7 and PAI-1 are indicated by arrows. The asterisk inSmad7 blot indicates the transfected F-Smad7 mRNA. The amount andintactness of total RNA loaded was checked by ethidium bromide staining(C and F). For junB (A) and Smad7 probe (B) the same blot was used,which was different from blot for PAI-11 probe (E). After treatment ofcells with zinc chloride for 20 h prior to TGF-β1 stimulation for 2 h,the ectopically induced Smad7 was found to inhibit TGF-β1-inducedimmediate early gene responses at a Smad7 mRNA extopic expression levelsimilar to that of endogenous gene after TGF-β treatment (FIG. 17B).Interestingly, upon treatment with zinc chloride for 6 h, no significanteffect on TGF-β1 stimulation of early response genes in the Smad7expressing clones was observed, whereas the apparent ectopic Smad7 mRNAlevels were higher after 6 h versus 20 h pretreatment. Possiblydifferences in Smad7 mRNA do not correlate exactly with Smad7 proteinlevels upon zinc chloride treatment, or a particularpost-translationally modification that occurs with slow kinetics may berequired for interaction of Smad7 with receptor in MV1Lu cells.

Example 19

TGF-β1-induced Nuclear Export of Smad7 in Transfected COS Cells andNontransfected Mv1Lu Cells

The subcellular localization of F-Smad6, F-Smad7 and F-Smad2 intransfected COS cells was analyzed in the absence or presence of theconstitutive active TβR-I(T204D) by immunofluorescence using Flagantibody (FIG. 18A). In FIG. 18, Panel A shows the subcellulardistribution of F-Smad7, F-Smad7 and F-Smad2 in the absence or presenceof constitutive TβR-I(T204D) mutant in transfected COS cells. Smads werelocalized in the cells by immunofluorescence using Flag antibody. PanelB shows the quantitation of nuclear versus cytoplasmic cell staining forF-smads in the absence or presence of T βR-I(T204D). Panel C shows thesubcellular distribution of Smad7 and Smad6 in nontransfected MV1Lucells in the absence and presence of TGF-β1. The Smads were localized inthe cells using specific antisera to Smad6 (ESP) or Smad7 (KER). Smad6was mainly located in the cytoplasm of the cells and the subcellularlocalization was not different from Smad6 cotransfection with TβR-I(T204D), constitutively active TβR-1. Smad7 was located in the cellnuclei but exported in a large proportion of total treated cells fromthe nuclei to the cytoplasm upon cotransfection with T βR-I(T204D),which is in agreement with earlier reports (Eppert et al. 1996; Nakao etal., 1997). The quantitation of nuclear versus cytoplasmic staining forthe different Smads in the absence or presence of TβR-I(T204D) arepresented in FIG. 18B. The nuclear localization was checked by DAPIstaining. The observed differences in subcellular distribution of Smad7versus Smad2 and Smad6, suggest a functional significance ofTβR-1-mediated export of Smad7 from the nucleus. However, overexpression in COS cells and use of epitope tag may affect Smad7subcellular distribution. Therefore, the subcellular distribution ofinhibitory Smads in nontransfected Mv1Lu cells using antisera thatspecifically recognize Smad6 and Smad7 was also examined. In agreementwith results in transfected COS cells, a cytoplasmic staining for Smad6in the absence or presence of TGF-β (or BMP-7) was observed. In someexperiments, a TGF-β1-induced nuclear translocation was observed in someof the cells (FIG. 18C). In the absence of ligand, a nuclear stainingwas observed for Smad7. However, upon TGF-β1 stimulation a cytoplasmicaccumulation of Smad7 was induced (FIG. 18C). Two different antisera foreach of Smad6 (recognizing Smad6L but not Smad6S) and Smad7, raisedagainst different epitopes, gave identical results.

Example 20

Differential Localization of Smad7 Deletion Mutants in the Absence orPresence of TGF-β1

The subcellular localization of F-Smad6 and F-Smad7 in Mv1Lu cellsstably expressing F-Smad7 and F-Smad7 were analyzed in the absence orpresence TGF-β1 using the Flag antibody and immunofluorescence. In FIG.19, the subcellular distribution of Smad7 mutants in the absence orpresence of TGF-β1 is shown. The cells were pretreated with zincchloride. Smads were localized in the cells using Flag antibody.Identical results were obtained, compared to the experiments in whichtransfected COS cells and nontransfected Mv1Lu cells were used;cytoplasmic staining for F-Smad6L and Smad6S versus a TGF-β1-inducednuclear export for F-Smad7 (FIG. 19). To gain more insight into regionsin Smad7 that are important for nuclear localization and TGF-β1-inducednuclear export, the subcellular distribution of different Smad7 deletionmutants were analyzed. pMEP4 expression constructs for the C-terminaldomain of F-Smad7 (7C; amino acids 204-426). F-Smad7C with C-taildeletion (7CΔ; amino acids 204-407) Smad7 with deletion of C-tail (7Δ;amino acids 1-407), and N-terminal domain of Smad7 (7N; amino acids1-203) were stably transfected into Mv1Lu cells, and cell linescharacterized for Smad7 expression upon zinc chloride treatment (FIG.15). All cells showed some degree of leaky expression, but in all celllines zinc treatment included expression of the Smad protein.Subcellular distribution of the 7C mutant (after zinc chloridetreatment) in the absence or presence of TGF-β1 was similar to that ofwild-type Smad7, although its efficiency of nuclear export was much lessthan that of wild-type Smad7. An intact MH2 domain appeared importantfor nuclear localization as 7CΔ and 7Δ mutants were predominantly incytoplasm in the absence of TGF-β1. Smad7N was found in the cytoplasm inthe absence or presence of TGF-β1; it had a spotted localizationsuggesting association with a particular cell structure (FIG. 19). Noneof the Smad7 deletion constructs were able to interfere withTGF-β1-induced growth inhibition as observed for Smad7 wild-type.

Example 21

Characterization of the Mouse Smad7 Promoter

The mouse Smad7 promoter was isolated from a commercially availablegenomic DNA library of the 129/Sv mouse strain (Stratagene, La Jolla,Calif.), using a ˜800 bp Smad7 cDNA fragment as a probe (nucleotides883-1614 of SEQ ID NO. 3).

The putative Smad7 promoter that was isolated comprised of a ˜4,465 bpXho I-Xho I fragment from the 5′-end of the Smad7 mouse genomic DNA. Asingle Bam HI site was identified by restriction mapping ˜3,840 bp fromthe 5′-Xho I site. In order to determine the promoter properties of the˜4,465 bp Xho I-Xho I DNA, three different regions of this DNA fragment(i.e., ˜4,465 bp Xho I-Xho I, ˜725 bp Bam HI-Xho I, and ˜3,840 bp XhoI-Bam HI) were inserted 5′ of the promoter-less luciferase reporter genein the commercially available pGL3-Basic expression vector (Promega,Madison, Wis.) and transfected into HepG2 cells. Following transfection,the cells were starved overnight in DMEM (0.3% FBS) and later stimulatedwith 10 ng/ml of TGF-β1 or vehicle (negative control) for 16 hours. Thecells were lysed and subjected to luciferase analysis. These results,depicted in FIG. 21, show that both the ˜4,465 bp Xho I-Xho I and the˜725 bp Bam HI-Xho I Smad7 DNA fragments but not the ˜3,840 bp Xho I-BamHI fragment, confer TGF-β1 inducibility to the heterologous luciferasetranscription unit.

The 725 bp Bam HI-Xho I Smad7 promoter fragment was furthercharacterized. Its sequence is depicted as SEQ ID NO. 15 of the sequencelisting. The transcriptional start site was localized by an RNAasePretection Assay and was localized at ˜nucleotide 499 of SEQ ID NO. 15.This start site was also found to coincide with the predicted start siteas determined by computer software programs searching for such sites(Wingender, E. et al., Biotechnology, 1994, 35:273-280; Prestidge, D. etal., J Mol Biol, 1995, 249:923-932). FIG. 22 depicts several restrictionenzyme sites and putative binding sites for different transcriptionfactors and Smad proteins in the Bam HI-Xho I promoter fragment. Theputative binding sites for non-Smad transcription factors wereidentified using the well known in the art TFSEARCH computer program andthe TRANSFAC MATRIX TABLE database. Putative Smad binding regions wereassessed using sequence information from the literature.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

A Sequence Listing is presented followed by what is claimed:

15 480 base pairs nucleic acid single linear Other not provided 1CAGCAACCAT GGACGGGTTT CACCGTGCAG ATCAGCTTTG TGAAGGGCTG GGGCCAGTGC 60TACACCCGCC AGTTCATCAG CAGCTGCCCG TGCTGGCTGG AGGTCATCTT CAACAGCCGG 120TAGTCGGTCG TGTGGTGGGG AGAAGAGGAC AGGGCGGATC GTGAGCCGAG CAGGCCACCG 180TTCAAACTAC TTGCTGCTAA CCTTTCCCGA GTGATTGCTT TTCATGCAAA CTCTTTGGTT 240GGTGTTGTTA TTGCCATTCA TTGTTGGTTT TGTTTTGTTC TGTTCTGGGT TCAGCCTGGC 300CTGCCCAGCC CTGGTCATCC AGCTGTACTG GCCCCTGGGG GGGTTCTGAG CAGTGCCTCT 360TGTCTTGGAG ACAGACCTGG TGCCTGGCGC TGCCTTCAGC AGGCAGCAGG CAGCCTCCTG 420CACACTGGCT TTTTTAGTCA TTTATGGGCA AAAAGAGTTA AAGTAAAACT TTGCAAATCC 480295 base pairs nucleic acid single linear not provided 2 ACTCTAGTTCACAGAGTCGA CTAAGGTGAT GGGGGTTGCA GCACACCAGC TCGGGGTTGA 60 TCTTCCCGTAAGATTCACAG CAACACAGCC TCTTGACTTC CGAGGAATGC CTGAGATCCG 120 GCCACCTGAACACTTTGCAC AGCAGGAGGG GGAGCGAGTA GGACGAGGGC GGCTGCACTG 180 GCTGCCGCTGGCGGGCACGC CCGGGCCCAG CCTGCAGTCC AGGCGGCCGG GCAGCAGGAG 240 GCACGCGGTGCGCGTACCGC CGCGGGACTC CACGGCCTGA AGCAGCAGCT CCAGC 295 1945 base pairsnucleic acid single linear not provided Coding Sequence 308...1585 3CGTTCCTCCA GGGTCACGCC GGGGCCCGAA AGCCGCGCAG GGCGCGGGCC GCGCCGGGTG 60GGGCATCCGA AGCGCAGCCC CCCGATCCCC GGCAGGCGCC CTGGGCCCCC GCGCGCGCCC 120CGGCCTCTGG GAGATGGCGC ATGCCAGGAG GGCCCCTCCG GCCGCCGCCG GTTCTGCCCG 180GGCCCCTGCT GTTGCTGCTG TCGCCTGCGC CTGNTGCCCC AAGTCGGCGC CCGATTNTTC 240ATGGTGTGCG GAGGTCATGT TCGCTCCTTA GCCGGCAAAC GATTTTCTCC TCGCCTCCTC 300GCCCCGC ATG TTC AGG ACC AAA CGA TCT GCG CTC GTC CGG CGT CTC TGG 349 MetPhe Arg Thr Lys Arg Ser Ala Leu Val Arg Arg Leu Trp 1 5 10 AGG AGC CGTGCG CCC GGC GGC GAG GAC GAG GAG GAG GGC GTG GGG GGT 397 Arg Ser Arg AlaPro Gly Gly Glu Asp Glu Glu Glu Gly Val Gly Gly 15 20 25 30 GGC GGC GGAGGA GGC GAG CTG CGG GGA GAA GGG GCG ACG GAC GGC CGG 445 Gly Gly Gly GlyGly Glu Leu Arg Gly Glu Gly Ala Thr Asp Gly Arg 35 40 45 GCT TAT GGG GCTGGT GGC GGC GGT GCG GGC AGG GCT GGC TGC TGC CTG 493 Ala Tyr Gly Ala GlyGly Gly Gly Ala Gly Arg Ala Gly Cys Cys Leu 50 55 60 GGC AAG GCA GTC CGAGGT GCC AAA GGT CAC CAC CAT CCC CAT CCC CCA 541 Gly Lys Ala Val Arg GlyAla Lys Gly His His His Pro His Pro Pro 65 70 75 ACC TCG GGT GCC GGG GCGGCC GGG GGC GCC GAG GCG GAT CTG AAG GCG 589 Thr Ser Gly Ala Gly Ala AlaGly Gly Ala Glu Ala Asp Leu Lys Ala 80 85 90 CTC ACG CAC TCG GTG CTC AAGAAA CTC AAG GAG CGG CAG CTG GAG CTG 637 Leu Thr His Ser Val Leu Lys LysLeu Lys Glu Arg Gln Leu Glu Leu 95 100 105 110 CTG CTT CAG GCC GTG GAGTCC CGC GGC GGT ACG CGC ACC GCG TGC CTC 685 Leu Leu Gln Ala Val Glu SerArg Gly Gly Thr Arg Thr Ala Cys Leu 115 120 125 CTG CTG CCC GGC CGC CTGGAC TGC AGG CTG GGC CCG GGG GCG CCC GCC 733 Leu Leu Pro Gly Arg Leu AspCys Arg Leu Gly Pro Gly Ala Pro Ala 130 135 140 AGC GCG CAG CCC GCG CAGCCG CCC TCG TCC TAC TCG CTC CCC CTC CTG 781 Ser Ala Gln Pro Ala Gln ProPro Ser Ser Tyr Ser Leu Pro Leu Leu 145 150 155 CTG TGC AAA GTG TTC AGGTGG CCG GAT CTC AGG CAT TCC TCG GAA GTC 829 Leu Cys Lys Val Phe Arg TrpPro Asp Leu Arg His Ser Ser Glu Val 160 165 170 AAG AGG CTG TGT TGC TGTGAA TCT TAC GGG AAG ATC AAC CCC GAG CTG 877 Lys Arg Leu Cys Cys Cys GluSer Tyr Gly Lys Ile Asn Pro Glu Leu 175 180 185 190 GTG TGC TGC AAC CCCCAT CAC CTT AGT CGA CTC TGT GAA CTA GAG TCT 925 Val Cys Cys Asn Pro HisHis Leu Ser Arg Leu Cys Glu Leu Glu Ser 195 200 205 CCC CCT CCT CCT TACTCC AGA TAC CCA ATG GAT TTT CTC AAA CCA ACT 973 Pro Pro Pro Pro Tyr SerArg Tyr Pro Met Asp Phe Leu Lys Pro Thr 210 215 220 GCA GGC TGT CCA GATGCT GTA CCT TCC TCC GCG GAA ACC GGG GGA ACG 1021 Ala Gly Cys Pro Asp AlaVal Pro Ser Ser Ala Glu Thr Gly Gly Thr 225 230 235 AAT TAT CTG GCC CCTGGG GGG CTT TCA GAT TCC CAA CTT CTT CTG GAG 1069 Asn Tyr Leu Ala Pro GlyGly Leu Ser Asp Ser Gln Leu Leu Leu Glu 240 245 250 CCT GGG GAT CGG TCACAC TGG TGC GTG GTG GCA TAC TGG GAG GAG AAG 1117 Pro Gly Asp Arg Ser HisTrp Cys Val Val Ala Tyr Trp Glu Glu Lys 255 260 265 270 ACT CGC GTG GGGAGG CTC TAC TGT GTC CAA GAG CCC TCC CTG GAT ATC 1165 Thr Arg Val Gly ArgLeu Tyr Cys Val Gln Glu Pro Ser Leu Asp Ile 275 280 285 TTC TAT GAT CTACCT CAG GGG AAT GGC TTT TGC CTC GGA CAG CTC AAT 1213 Phe Tyr Asp Leu ProGln Gly Asn Gly Phe Cys Leu Gly Gln Leu Asn 290 295 300 TCG GAC AAC AAGAGT CAG CTG GTA CAG AAA GTG CGG AGC AAG ATC GGC 1261 Ser Asp Asn Lys SerGln Leu Val Gln Lys Val Arg Ser Lys Ile Gly 305 310 315 TGT GGC ATC CAGCTG ACG CGG GAA GTG GAT GGC GTG TGG GTT TAC AAC 1309 Cys Gly Ile Gln LeuThr Arg Glu Val Asp Gly Val Trp Val Tyr Asn 320 325 330 CGC AGC AGT TACCCC ATC TTC ATC AAG TCC GCC ACA CTG GAC AAC CCG 1357 Arg Ser Ser Tyr ProIle Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro 335 340 345 350 GAC TCC AGGACG CTG TTG GTG CAC AAA GTG TTC CCT GGT TTC TCC ATC 1405 Asp Ser Arg ThrLeu Leu Val His Lys Val Phe Pro Gly Phe Ser Ile 355 360 365 AAG GCT TTTGAC TAT GAG AAA GCC TAC AGC CTG CAG CGG CCC AAT GAC 1453 Lys Ala Phe AspTyr Glu Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp 370 375 380 CAC GAG TTCATG CAG CAA CCA TGG ACG GGT TTC ACC GTG CAG ATC AGC 1501 His Glu Phe MetGln Gln Pro Trp Thr Gly Phe Thr Val Gln Ile Ser 385 390 395 TTT GTG AAGGGC TGG GGC CAG TGC TAC ACC CGC CAG TTC ATC AGC AGC 1549 Phe Val Lys GlyTrp Gly Gln Cys Tyr Thr Arg Gln Phe Ile Ser Ser 400 405 410 TGC CCG TGCTGG CTG GAG GTC ATC TTC AAC AGC CGG TAGTCGGTCG TGTGGT 1601 Cys Pro CysTrp Leu Glu Val Ile Phe Asn Ser Arg 415 420 425 GGGGAGAAGA GGACAGGGCGGATCGTGAGC CGAGCAGGCC ACCGTTCAAA CTACTTGCTG 1661 CTAACCTTTC CCGAGTGATTGCTTTTCATG CAAACTCTTT GGTTGGTGTT GTTATTGCCA 1721 TTCATTGTTG GTTTTGTTTTGTTCTGTTCT GGGTTCAGCC TGGCCTGCCC AGCCCTGGTC 1781 ATCCAGCTGT ACTGGCCCCTGGGGGGGTTC TGAGCAGTGC CTCTTGTCTT GGAGACAGAC 1841 CTGGTGCCTG GCGCTGCCTTCAGCAGGCAG CAGGCAGCCT CCTGCACACT GGCTTTTTTA 1901 GTCATTTATG GGCAAAAAGAGTTAAAGTAA AACTTTGCAA ATCC 1945 426 amino acids amino acid single linearprotein internal not provided 4 Met Phe Arg Thr Lys Arg Ser Ala Leu ValArg Arg Leu Trp Arg Ser 1 5 10 15 Arg Ala Pro Gly Gly Glu Asp Glu GluGlu Gly Val Gly Gly Gly Gly 20 25 30 Gly Gly Gly Glu Leu Arg Gly Glu GlyAla Thr Asp Gly Arg Ala Tyr 35 40 45 Gly Ala Gly Gly Gly Gly Ala Gly ArgAla Gly Cys Cys Leu Gly Lys 50 55 60 Ala Val Arg Gly Ala Lys Gly His HisHis Pro His Pro Pro Thr Ser 65 70 75 80 Gly Ala Gly Ala Ala Gly Gly AlaGlu Ala Asp Leu Lys Ala Leu Thr 85 90 95 His Ser Val Leu Lys Lys Leu LysGlu Arg Gln Leu Glu Leu Leu Leu 100 105 110 Gln Ala Val Glu Ser Arg GlyGly Thr Arg Thr Ala Cys Leu Leu Leu 115 120 125 Pro Gly Arg Leu Asp CysArg Leu Gly Pro Gly Ala Pro Ala Ser Ala 130 135 140 Gln Pro Ala Gln ProPro Ser Ser Tyr Ser Leu Pro Leu Leu Leu Cys 145 150 155 160 Lys Val PheArg Trp Pro Asp Leu Arg His Ser Ser Glu Val Lys Arg 165 170 175 Leu CysCys Cys Glu Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys 180 185 190 CysAsn Pro His His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro 195 200 205Pro Pro Tyr Ser Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Gly 210 215220 Cys Pro Asp Ala Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr 225230 235 240 Leu Ala Pro Gly Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu ProGly 245 250 255 Asp Arg Ser His Trp Cys Val Val Ala Tyr Trp Glu Glu LysThr Arg 260 265 270 Val Gly Arg Leu Tyr Cys Val Gln Glu Pro Ser Leu AspIle Phe Tyr 275 280 285 Asp Leu Pro Gln Gly Asn Gly Phe Cys Leu Gly GlnLeu Asn Ser Asp 290 295 300 Asn Lys Ser Gln Leu Val Gln Lys Val Arg SerLys Ile Gly Cys Gly 305 310 315 320 Ile Gln Leu Thr Arg Glu Val Asp GlyVal Trp Val Tyr Asn Arg Ser 325 330 335 Ser Tyr Pro Ile Phe Ile Lys SerAla Thr Leu Asp Asn Pro Asp Ser 340 345 350 Arg Thr Leu Leu Val His LysVal Phe Pro Gly Phe Ser Ile Lys Ala 355 360 365 Phe Asp Tyr Glu Lys AlaTyr Ser Leu Gln Arg Pro Asn Asp His Glu 370 375 380 Phe Met Gln Gln ProTrp Thr Gly Phe Thr Val Gln Ile Ser Phe Val 385 390 395 400 Lys Gly TrpGly Gln Cys Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro 405 410 415 Cys TrpLeu Glu Val Ile Phe Asn Ser Arg 420 425 1876 base pairs nucleic acidsingle linear not provided Coding Sequence 50...1327 5 GAATTCGGCACGAGGGCAAA CGACTTTTCT CCTCGCCTCC TCGCCCCGC ATG TTC AGG 58 Met Phe Arg 1ACC AAA CGA TCT GCG CTC GTC CGG CGT CTC TGG AGG AGC CGT GCG CCC 106 ThrLys Arg Ser Ala Leu Val Arg Arg Leu Trp Arg Ser Arg Ala Pro 5 10 15 GGCGGC GAG GAC GAG GAG GAG GGC GCA GGG GGA GGT GGA GGA GGA GGC 154 Gly GlyGlu Asp Glu Glu Glu Gly Ala Gly Gly Gly Gly Gly Gly Gly 20 25 30 35 GAGCTG CGG GGA GAA GGG GCG ACG GAC AGC CGA GCG CAT GGG GCC GGT 202 Glu LeuArg Gly Glu Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly 40 45 50 GGC GGCGGC CCG GGC AGG GCT GGA TGC TGC CTG GGC AAG GCG GTG CGA 250 Gly Gly GlyPro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg 55 60 65 GGT GCC AAATGT CAC CAC CAT CCC CAC CCG CCA GCC GCG GGC GCC GGC 298 Gly Ala Lys CysHis His His Pro His Pro Pro Ala Ala Gly Ala Gly 70 75 80 GCG GCC GGG GGCGCC GAG GCG GAT CTG AAG GCG CTC ACG CAC TCG GTG 346 Ala Ala Gly Gly AlaGlu Ala Asp Leu Lys Ala Leu Thr His Ser Val 85 90 95 CTC AAG AAA CTG AAGGAG CGG CAG CTG GAG CTG CTG CTC CAG GCC GTG 394 Leu Lys Lys Leu Lys GluArg Gln Leu Glu Leu Leu Leu Gln Ala Val 100 105 110 115 GAG TCC CGC GGCGGG ACG CGC ACC GCG TGC CTC CTG CTG CCC GGC CGC 442 Glu Ser Arg Gly GlyThr Arg Thr Ala Cys Leu Leu Leu Pro Gly Arg 120 125 130 CTG GAC TGC AGGCTG GGC CCG GGG GCG CCC GCC GGC GCG CAG CCT GCG 490 Leu Asp Cys Arg LeuGly Pro Gly Ala Pro Ala Gly Ala Gln Pro Ala 135 140 145 CAG CCG CCC TCGTCC TAC TCG CTC CCC CTC CTG CTG TGC AAA GTG TTC 538 Gln Pro Pro Ser SerTyr Ser Leu Pro Leu Leu Leu Cys Lys Val Phe 150 155 160 AGG TGG CCG GATCTC AGG CAT TCC TCG GAA GTC AAG AGG CTG TGT TGC 586 Arg Trp Pro Asp LeuArg His Ser Ser Glu Val Lys Arg Leu Cys Cys 165 170 175 TGT GAA TCT TACGGG AAG ATC AAC CCC GAG CTG GTG TGC TGC AAC CCC 634 Cys Glu Ser Tyr GlyLys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro 180 185 190 195 CAT CAC CTTAGC CGA CTC TGC GAA CTA GAG TCT CCC CCC CCT CCT TAC 682 His His Leu SerArg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr 200 205 210 TCC AGA TACCCG ATG GAT TTT CTC AAA CCA ACT GCA GAC TGT CCA GAT 730 Ser Arg Tyr ProMet Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro Asp 215 220 225 GCT GTG CCTTCC TCC GCT GAA ACA GGG GGA ACG AAT TAT CTG GCC CCT 778 Ala Val Pro SerSer Ala Glu Thr Gly Gly Thr Asn Tyr Leu Ala Pro 230 235 240 GGG GGG CTTTCA GAT TCC CAA CTT CTT CTG GAG CCT GGG GAT CGG TCA 826 Gly Gly Leu SerAsp Ser Gln Leu Leu Leu Glu Pro Gly Asp Arg Ser 245 250 255 CAC TGG TGCGTG GTG GCA TAC TGG GAG GAG AAG ACG AGA GTG GGG AGG 874 His Trp Cys ValVal Ala Tyr Trp Glu Glu Lys Thr Arg Val Gly Arg 260 265 270 275 CTC TACTGT GTC CAG GAG CCC TCT CTG GAT ATC TTC TAT GAT CTA CCT 922 Leu Tyr CysVal Gln Glu Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro 280 285 290 CAG GGGAAT GGC TTT TGC CTC GGA CAG CTC AAT TCG GAC AAC AAG AGT 970 Gln Gly AsnGly Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser 295 300 305 CAG CTGGTG CAG AAG GTG CGG AGC AAA ATC GGC TGC GGC ATC CAG CTG 1018 Gln Leu ValGln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu 310 315 320 ACG CGGGAG GTG GAT GGT GTG TGG GTG TAC AAC CGC AGC AGT TAC CCC 1066 Thr Arg GluVal Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro 325 330 335 ATC TTCATC AAG TCC GCC ACA CTG GAC AAC CCG GAC TCC AGG ACG CTG 1114 Ile Phe IleLys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr Leu 340 345 350 355 TTGGTA CAC AAG GTG TTC CCC GGT TTC TCC ATC AAG GCT TTC GAC TAC 1162 Leu ValHis Lys Val Phe Pro Gly Phe Ser Ile Lys Ala Phe Asp Tyr 360 365 370 GAGAAG GCG TAC AGC CTG CAG CGG CCC AAT GAC CAC GAG TTT ATG CAG 1210 Glu LysAla Tyr Ser Leu Gln Arg Pro Asn Asp His Glu Phe Met Gln 375 380 385 CAGCCG TGG ACG GGC TTT ACC GTG CAG ATC AGC TTT GTG AAG GGC TGG 1258 Gln ProTrp Thr Gly Phe Thr Val Gln Ile Ser Phe Val Lys Gly Trp 390 395 400 GGCCAG TGC TAC ACC CGC CAG TTC ATC AGC AGC TGC CCG TGC TGG CTA 1306 Gly GlnCys Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu 405 410 415 GAGGTC ATC TTC AAC AGC CGG TAGCCGCGTG CGGAGGGGAC AGAGCGTGAG CTGA 1361 GluVal Ile Phe Asn Ser Arg 420 425 GCAGGCCACA CTTCAAACTA CTTTGCTGCTAATATTTTCC TCCTGAGTGC TTGCTTTTCA 1421 TGCAAACTCT TTGGTCGTTT TTTTTTTGTTTGTTGGTTGG TTTTCTTCTT CTCGTCCTCG 1481 TTTGTGTTCT ATTTTTCTAA CTACAAAGGTTTAAATGAAC AAGAGAAGCA TCTCTCATTG 1541 GAAATTTAGC ATTGTAGTGC TTTGAGAGAGAAAGGACTCC CTGNAAAAAA ACCTGAGATT 1601 TATTAAAGNA AAAAATGTAT TTTATGTTATATATAAATAT ATTATTACTT GTAAATATAA 1661 AGACGTTTTA TAAGCATCAT TATTTATGTATTGTGCAATG TGTATAAACN AGNAAAATAA 1721 AGAAAAGATG CACTTTGCTT TAATATAAATGCAAATAACA AATGCCAAAT TAAAAAAGAT 1781 AAACACAAGA TTGGTGTTTT TTTCTATGGGTGTTATCACC TAGCNGAATG TTTTTCTAAA 1841 GGAGTTTATG TTCCATTAAA CGATTTTTAAAANGT 1876 426 amino acids amino acid single linear protein internal notprovided 6 Met Phe Arg Thr Lys Arg Ser Ala Leu Val Arg Arg Leu Trp ArgSer 1 5 10 15 Arg Ala Pro Gly Gly Glu Asp Glu Glu Glu Gly Ala Gly GlyGly Gly 20 25 30 Gly Gly Gly Glu Leu Arg Gly Glu Gly Ala Thr Asp Ser ArgAla His 35 40 45 Gly Ala Gly Gly Gly Gly Pro Gly Arg Ala Gly Cys Cys LeuGly Lys 50 55 60 Ala Val Arg Gly Ala Lys Cys His His His Pro His Pro ProAla Ala 65 70 75 80 Gly Ala Gly Ala Ala Gly Gly Ala Glu Ala Asp Leu LysAla Leu Thr 85 90 95 His Ser Val Leu Lys Lys Leu Lys Glu Arg Gln Leu GluLeu Leu Leu 100 105 110 Gln Ala Val Glu Ser Arg Gly Gly Thr Arg Thr AlaCys Leu Leu Leu 115 120 125 Pro Gly Arg Leu Asp Cys Arg Leu Gly Pro GlyAla Pro Ala Gly Ala 130 135 140 Gln Pro Ala Gln Pro Pro Ser Ser Tyr SerLeu Pro Leu Leu Leu Cys 145 150 155 160 Lys Val Phe Arg Trp Pro Asp LeuArg His Ser Ser Glu Val Lys Arg 165 170 175 Leu Cys Cys Cys Glu Ser TyrGly Lys Ile Asn Pro Glu Leu Val Cys 180 185 190 Cys Asn Pro His His LeuSer Arg Leu Cys Glu Leu Glu Ser Pro Pro 195 200 205 Pro Pro Tyr Ser ArgTyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp 210 215 220 Cys Pro Asp AlaVal Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr 225 230 235 240 Leu AlaPro Gly Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu Pro Gly 245 250 255 AspArg Ser His Trp Cys Val Val Ala Tyr Trp Glu Glu Lys Thr Arg 260 265 270Val Gly Arg Leu Tyr Cys Val Gln Glu Pro Ser Leu Asp Ile Phe Tyr 275 280285 Asp Leu Pro Gln Gly Asn Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp 290295 300 Asn Lys Ser Gln Leu Val Gln Lys Val Arg Ser Lys Ile Gly Cys Gly305 310 315 320 Ile Gln Leu Thr Arg Glu Val Asp Gly Val Trp Val Tyr AsnArg Ser 325 330 335 Ser Tyr Pro Ile Phe Ile Lys Ser Ala Thr Leu Asp AsnPro Asp Ser 340 345 350 Arg Thr Leu Leu Val His Lys Val Phe Pro Gly PheSer Ile Lys Ala 355 360 365 Phe Asp Tyr Glu Lys Ala Tyr Ser Leu Gln ArgPro Asn Asp His Glu 370 375 380 Phe Met Gln Gln Pro Trp Thr Gly Phe ThrVal Gln Ile Ser Phe Val 385 390 395 400 Lys Gly Trp Gly Gln Cys Tyr ThrArg Gln Phe Ile Ser Ser Cys Pro 405 410 415 Cys Trp Leu Glu Val Ile PheAsn Ser Arg 420 425 1281 base pairs nucleic acid single linear cDNA notprovided 7 ATGTTCAGGA CCAAACGATC TGCGCTCGTC CGGCGTCTCT GGAGGAGCCGTGCGCCCGGC 60 GGCGAGGACG AGGAGGAGGG CGTGGGGGGT GGCGGCGGAG GAGGCGAGCTGCGGGGAGAA 120 GGGGCGACGG ACGGCCGGGC TTATGGGGCT GGTGGCGGCG GTGCGGGCAGGGCTGGCTGC 180 TGCCTGGGCA AGGCAGTCCG AGGTGCCAAA GGTCACCACC ATCCCCATCCCCCAACCTCG 240 GGTGCCGGGG CGGCCGGGGG CGCCGAGGCG GATCTGAAGG CGCTCACGCACTCGGTGCTC 300 AAGAAACTCA AGGAGCGGCA GCTGGAGCTG CTGCTTCAGG CCGTGGAGTCCCGCGGCGGT 360 ACGCGCACCG CGTGCCTCCT GCTGCCCGGC CGCCTGGACT GCAGGCTGGGCCCGGGGGCG 420 CCCGCCAGCG CGCAGCCCGC GCAGCCGCCC TCGTCCTACT CGCTCCCCCTCCTGCTGTGC 480 AAAGTGTTCA GGTGGCCGGA TCTCAGGCAT TCCTCGGAAG TCAAGAGGCTGTGTTGCTGT 540 GAATCTTACG GGAAGATCAA CCCCGAGCTG GTGTGCTGCA ACCCCCATCACCTTAGTCGA 600 CTCTGTGAAC TAGAGTCTCC CCCTCCTCCT TACTCCAGAT ACCCAATGGATTTTCTCAAA 660 CCAACTGCAG GCTGTCCAGA TGCTGTACCT TCCTCCGCGG AAACCGGGGGAACGAATTAT 720 CTGGCCCCTG GGGGGCTTTC AGATTCCCAA CTTCTTCTGG AGCCTGGGGATCGGTCACAC 780 TGGTGCGTGG TGGCATACTG GGAGGAGAAG ACTCGCGTGG GGAGGCTCTACTGTGTCCAA 840 GAGCCCTCCC TGGATATCTT CTATGATCTA CCTCAGGGGA ATGGCTTTTGCCTCGGACAG 900 CTCAATTCGG ACAACAAGAG TCAGCTGGTA CAGAAAGTGC GGAGCAAGATCGGCTGTGGC 960 ATCCAGCTGA CGCGGGAAGT GGATGGCGTG TGGGTTTACA ACCGCAGCAGTTACCCCATC 1020 TTCATCAAGT CCGCCACACT GGACAACCCG GACTCCAGGA CGCTGTTGGTGCACAAAGTG 1080 TTCCCTGGTT TCTCCATCAA GGCTTTTGAC TATGAGAAAG CCTACAGCCTGCAGCGGCCC 1140 AATGACCACG AGTTCATGCA GCAACCATGG ACGGGTTTCA CCGTGCAGATCAGCTTTGTG 1200 AAGGGCTGGG GCCAGTGCTA CACCCGCCAG TTCATCAGCA GCTGCCCGTGCTGGCTGGAG 1260 GTCATCTTCA ACAGCCGGTA G 1281 1281 base pairs nucleicacid single linear cDNA not provided 8 ATGTTCAGGA CCAAACGATC TGCGCTCGTCCGGCGTCTCT GGAGGAGCCG TGCGCCCGGC 60 GGCGAGGACG AGGAGGAGGG CGCAGGGGGAGGTGGAGGAG GAGGCGAGCT GCGGGGAGAA 120 GGGGCGACGG ACAGCCGAGC GCATGGGGCCGGTGGCGGCG GCCCGGGCAG GGCTGGATGC 180 TGCCTGGGCA AGGCGGTGCG AGGTGCCAAATGTCACCACC ATCCCCACCC GCCAGCCGCG 240 GGCGCCGGCG CGGCCGGGGG CGCCGAGGCGGATCTGAAGG CGCTCACGCA CTCGGTGCTC 300 AAGAAACTGA AGGAGCGGCA GCTGGAGCTGCTGCTCCAGG CCGTGGAGTC CCGCGGCGGG 360 ACGCGCACCG CGTGCCTCCT GCTGCCCGGCCGCCTGGACT GCAGGCTGGG CCCGGGGGCG 420 CCCGCCGGCG CGCAGCCTGC GCAGCCGCCCTCGTCCTACT CGCTCCCCCT CCTGCTGTGC 480 AAAGTGTTCA GGTGGCCGGA TCTCAGGCATTCCTCGGAAG TCAAGAGGCT GTGTTGCTGT 540 GAATCTTACG GGAAGATCAA CCCCGAGCTGGTGTGCTGCA ACCCCCATCA CCTTAGCCGA 600 CTCTGCGAAC TAGAGTCTCC CCCCCCTCCTTACTCCAGAT ACCCGATGGA TTTTCTCAAA 660 CCAACTGCAG ACTGTCCAGA TGCTGTGCCTTCCTCCGCTG AAACAGGGGG AACGAATTAT 720 CTGGCCCCTG GGGGGCTTTC AGATTCCCAACTTCTTCTGG AGCCTGGGGA TCGGTCACAC 780 TGGTGCGTGG TGGCATACTG GGAGGAGAAGACGAGAGTGG GGAGGCTCTA CTGTGTCCAG 840 GAGCCCTCTC TGGATATCTT CTATGATCTACCTCAGGGGA ATGGCTTTTG CCTCGGACAG 900 CTCAATTCGG ACAACAAGAG TCAGCTGGTGCAGAAGGTGC GGAGCAAAAT CGGCTGCGGC 960 ATCCAGCTGA CGCGGGAGGT GGATGGTGTGTGGGTGTACA ACCGCAGCAG TTACCCCATC 1020 TTCATCAAGT CCGCCACACT GGACAACCCGGACTCCAGGA CGCTGTTGGT ACACAAGGTG 1080 TTCCCCGGTT TCTCCATCAA GGCTTTCGACTACGAGAAGG CGTACAGCCT GCAGCGGCCC 1140 AATGACCACG AGTTTATGCA GCAGCCGTGGACGGGCTTTA CCGTGCAGAT CAGCTTTGTG 1200 AAGGGCTGGG GCCAGTGCTA CACCCGCCAGTTCATCAGCA GCTGCCCGTG CTGGCTAGAG 1260 GTCATCTTCA ACAGCCGGTA G 1281 1491base pairs nucleic acid single linear cDNA not provided 9 ATGTTCAGGTCCAAACGCTC GGGGCTGGTG CGGCGACTTT GGCGAAGTCG TGTGGTCCCC 60 GACCGGGAGGAAGGCGGCAG CGGCGGCGGC GGTGGCGGCG ACGAGGATGG GAGCTTGGGC 120 AGCCGAGCTGAGCCGGCCCC GCGGGCAAGA GAGGGCGGAG GCTGCGGCCG CTCCGAAGTC 180 CGCCCGGTAGCCCCGCGGCG GCCCCGGGAC GCAGTGGGAC AGCGAGGCGC CCAGGGCGCG 240 GGGAGGCGCCGGCGCGCAGG GGGCCCCCCG AGGCCCATGT CGGAGCCAGG GGCCGGCGCT 300 GGGAGCTCCCTGCTGGACGT GGCGGAGCCG GGAGGCCCGG GCTGGCTGCC CGAGAGTGAC 360 TGCGAGACGGTGACCTGCTG TCTCTTTTCG GAGCGGGACG CCGCCGGCGC GCCCCGGGAC 420 GCCAGCGACCCCCTGGCCGG GGCGGCCCTG GAGCCGGCGG GCGGCGGGCG GAGTCGCGAA 480 GCGCGCTCGCGGCTGCTGCT GCTGGAGCAG GAACTCAAAA CCGTCACGTA CTCGCTGCTG 540 AAGCGGCTCAAGGAGCGCTC GCTGGACACG CTGCTGGAGG CGGTGGAGTC CCGCGGCGGC 600 GTGCCGGGCGGCTGCGTGCT GGTGCCGCGC GCCGACCTCC GCCTGGGCGG CCAGCCCGCG 660 CCGCCGCAGCTGCTGCTCGG CCGCCTCTTT CGCTGGCCCG ACCTGCAGCA CGCCGTGGAG 720 CTGAAGCCCCTGTGCGGCTG CCACAGCTTC GCCGCCGCCG CCGACGGCCC TACCGTGTGC 780 TGCAACCCCTACCACTTCAG CCGGCTCTGC GGGCCCGAAT CTCCGCCACC TCCCTACTCT 840 CGGCTGTCTCCTCGCGACGA GTACAAGCCA CTGGATCTGT CCGATTCCAC ATTGTCTTAC 900 ACTGAAACGGAGGCTACCAA CTCCCTCATC ACTGCTCCGG GTGAATTCTC AGACGCCAGC 960 ATGTCTCCGGACGCCACCAA GCCGAGCCAC TGGTGCAGCG TGGCGTACTG GGAGCACCGG 1020 ACGCGCGTGGGCCGCCTCTA TGCGGTGTAC GACCAGGCCG TCAGCATCTT CTACGACCTA 1080 CCTCAGGGCAGCGGCTTCTG CCTGGGCCAG CTCAACCTGG AGCAGCGCAG CGAGTCGGTG 1140 CGGCGAACGCGCAGCAAGAT CGGCTTCGGC ATCCTGCTCA GCAAGGAGCC CGACGGCGTG 1200 TGGGCCTACAACCGCGGCGA GCACCCCATC TTCGTCAACT CCCCGACGCT GGACGCGCCC 1260 GGCGGCCGCGCCCTGGTCGT GCGCAAGGTG CCCCCCGGCT ACTCCATCAA GGTGTTCGAC 1320 TTCGAGCGCTCGGGCCTGCA GCACGCGCCC GAGCCCGACG CCGCCGACGG CCCCTACGAC 1380 CCCAACAGCGTCCGCATCAG CTTCGCCAAG GGCTGGGGGC CCTGCTACTC CCGGCAGTTC 1440 ATCACCTCCTGCCCCTGCTG GCTGGAGATC CTCCTCAACA ACCCCAGATA G 1491 496 amino acids aminoacid single linear protein not provided 10 Met Phe Arg Ser Lys Arg SerGly Leu Val Arg Arg Leu Trp Arg Ser 1 5 10 15 Arg Val Val Pro Asp ArgGlu Glu Gly Gly Ser Gly Gly Gly Gly Gly 20 25 30 Gly Asp Glu Asp Gly SerLeu Gly Ser Arg Ala Glu Pro Ala Pro Arg 35 40 45 Ala Arg Glu Gly Gly GlyCys Gly Arg Ser Glu Val Arg Pro Val Ala 50 55 60 Pro Arg Arg Pro Arg AspAla Val Gly Gln Arg Gly Ala Gln Gly Ala 65 70 75 80 Gly Arg Arg Arg ArgAla Gly Gly Pro Pro Arg Pro Met Ser Glu Pro 85 90 95 Gly Ala Gly Ala GlySer Ser Leu Leu Asp Val Ala Glu Pro Gly Gly 100 105 110 Pro Gly Trp LeuPro Glu Ser Asp Cys Glu Thr Val Thr Cys Cys Leu 115 120 125 Phe Ser GluArg Asp Ala Ala Gly Ala Pro Arg Asp Ala Ser Asp Pro 130 135 140 Leu AlaGly Ala Ala Leu Glu Pro Ala Gly Gly Gly Arg Ser Arg Glu 145 150 155 160Ala Arg Ser Arg Leu Leu Leu Leu Glu Gln Glu Leu Lys Thr Val Thr 165 170175 Tyr Ser Leu Leu Lys Arg Leu Lys Glu Arg Ser Leu Asp Thr Leu Leu 180185 190 Glu Ala Val Glu Ser Arg Gly Gly Val Pro Gly Gly Cys Val Leu Val195 200 205 Pro Arg Ala Asp Leu Arg Leu Gly Gly Gln Pro Ala Pro Pro GlnLeu 210 215 220 Leu Leu Gly Arg Leu Phe Arg Trp Pro Asp Leu Gln His AlaVal Glu 225 230 235 240 Leu Lys Pro Leu Cys Gly Cys His Ser Phe Ala AlaAla Ala Asp Gly 245 250 255 Pro Thr Val Cys Cys Asn Pro Tyr His Phe SerArg Leu Cys Gly Pro 260 265 270 Glu Ser Pro Pro Pro Pro Tyr Ser Arg LeuSer Pro Arg Asp Glu Tyr 275 280 285 Lys Pro Leu Asp Leu Ser Asp Ser ThrLeu Ser Tyr Thr Glu Thr Glu 290 295 300 Ala Thr Asn Ser Leu Ile Thr AlaPro Gly Glu Phe Ser Asp Ala Ser 305 310 315 320 Met Ser Pro Asp Ala ThrLys Pro Ser His Trp Cys Ser Val Ala Tyr 325 330 335 Trp Glu His Arg ThrArg Val Gly Arg Leu Tyr Ala Val Tyr Asp Gln 340 345 350 Ala Val Ser IlePhe Tyr Asp Leu Pro Gln Gly Ser Gly Phe Cys Leu 355 360 365 Gly Gln LeuAsn Leu Glu Gln Arg Ser Glu Ser Val Arg Arg Thr Arg 370 375 380 Ser LysIle Gly Phe Gly Ile Leu Leu Ser Lys Glu Pro Asp Gly Val 385 390 395 400Trp Ala Tyr Asn Arg Gly Glu His Pro Ile Phe Val Asn Ser Pro Thr 405 410415 Leu Asp Ala Pro Gly Gly Arg Ala Leu Val Val Arg Lys Val Pro Pro 420425 430 Gly Tyr Ser Ile Lys Val Phe Asp Phe Glu Arg Ser Gly Leu Gln His435 440 445 Ala Pro Glu Pro Asp Ala Ala Asp Gly Pro Tyr Asp Pro Asn SerVal 450 455 460 Arg Ile Ser Phe Ala Lys Gly Trp Gly Pro Cys Tyr Ser ArgGln Phe 465 470 475 480 Ile Thr Ser Cys Pro Cys Trp Leu Glu Ile Leu LeuAsn Asn Pro Arg 485 490 495 20 amino acids amino acid single linearpeptide not provided 11 Glu Ser Pro Pro Pro Pro Tyr Ser Arg Leu Ser ProArg Asp Glu Tyr 1 5 10 15 Lys Pro Leu Asp 20 18 amino acids amino acidsingle linear peptide not provided 12 Ser Arg Gln Phe Ile Thr Ser CysPro Cys Trp Leu Glu Ile Leu Asn 1 5 10 15 Pro Arg 21 amino acids aminoacid single linear peptide not provided 13 Lys Glu Arg Gln Leu Glu LeuLeu Leu Gln Ala Val Glu Ser Arg Gly 1 5 10 15 Gly Thr Arg Thr Ala 20 15amino acids amino acid single linear peptide not provided 14 Lys Ala ValArg Gly Ala Lys Gly His His His Pro His Pro Pro 1 5 10 15 733 base pairsnucleic acid single linear not provided 15 CTCGAGATCC GCCGCCAGGGCCTTAAAACA AAACGAATGA ATGAAGCGCC CGGGCGAGCT 60 CTGCTGGTCT AAATCGGGCCACTAACCGGC TCATTCCGCT CCAAACTCGC CCGCTCCATG 120 CCCCCAGCTC TTCCGATTTCCCCCTGCTCC CGCTGGGTCT CCGCCAGACC CCCGGGGCCG 180 TCTCGGCCTC CCACCTCCCTCCACCGTGGG TACCCCTCTC TAGACCTGGG AGAGGGTGGC 240 AGTAACTGGG AGGGGGGTTGAAATAGCTTT TAGAAACCCG ATCTGTTGTT TGCGAAACAC 300 AATCGCTTTT TTTTTTTTTAAAGCGACAGG GTGTCTAGAC GGCCACGTGA CGAGGCCGGA 360 GCCGGGCGCG CCACTGCGCAGTGGAACCAG CCGAGCAGAG GGACGGGTGG GGGGGGCGGG 420 AAGGAGGCGG CGGCGGCTGGGGGCGGGGGA GGGAGGGGTA GAGGGGGGAG GGAAGGGGGC 480 GGAGGCGGGA GGCCTTGCGGGAGGCGGCGA GCCCTGGGCA CATTCGCTCG CTGATCGGCG 540 CACAGAGGAT CTTGTCCCCGAGCTGCGCCA GCAGAGCCAG CCGGGCGCCT CGCTCGGTCC 600 GCTCGCCGCG CCGGAGAGAGCTGCCTGAGA CGCAGCCAGC CAGCCAGCCG GCGCCACGCC 660 GCCGAGCGCT CGGCCCCGGAGTCCCTGAGT GCGGCGCGGC GAGCCCCCAG CGGCGGCAGA 720 AGGACTCGAG ATC 733

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of (a) nucleic acid molecules comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7 and SEQ ID NO:8, and (b) full-length complements of(a).
 2. The isolated nucleic acid molecule of claim 1, wherein theisolated nucleic acid molecule comprises SEQ ID NO:7 or SEQ ID NO:8. 3.The isolated nucleic acid molecule of claim 1, wherein the isolatednucleic acid molecule consists of SEQ ID NO:3.
 4. The isolated nucleicacid molecule of claim 1, wherein the isolated nucleic acid moleculeconsists of SEQ ID NO:5.
 5. An expression vector comprising the isolatednucleic acid molecule of claim 1 operably linked to a promoter.
 6. Ahost cell transformed or transfected with the expression vector of claim5.
 7. The isolated nucleic acid molecule of claim 2, wherein theisolated nucleic acid molecule consists of SEQ ID NO:7.
 8. The isolatednucleic acid molecule of claim 2, wherein the isolated nucleic acidmolecule consists of SEQ ID NO:8.
 9. A method for making a Smad7polypeptide comprising culturing the host cell of claim 6 in a culturemedium and isolating the polypeptide from the host cell or culturemedium.
 10. A method for making a Smad7 polypeptide comprisingintroducing the nucleic acid molecule of claim 1 into a non-cell systemfor transcription and/or translation of a nucleic acid molecule,incubating the non-cell system under conditions sufficient fortranscription and/or translation of the nucleic acid molecule andisolating the polypeptide produced by the transcription and/ortranslation from the non-cell system.
 11. A method for making a Smad7polypeptide comprising introducing the expression vector of claim 5 intoa non-cell system for transcription and/or translation of a nucleic acidmolecule, incubating the non-cell system under conditions sufficient fortranscription and/or translation of the nucleic acid molecule andisolating the polypeptide produced by the transcription and/ortranslation from the non-cell system.
 12. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding an amino acidsequence selected from the group consisting of SEQ ID NO:4 and SEQ IDNO:6.
 13. An isolated nucleic acid molecule consisting of a nucleotidesequence encoding an amino acid sequence selected from the groupconsisting of SEQ ID NO:4 and SEQ ID NO:6.
 14. An expression vectorcomprising the isolated nucleic acid molecule of claim 12 operablylinked to a promoter.
 15. A host cell transformed or transfected withthe expression vector of claim
 14. 16. A method for making a Smad7polypeptide comprising culturing the host cell of claim 15 in a culturemedium and isolating the polypeptide from the host cell or culturemedium.
 17. A method for making a Smad7 polypeptide comprisingintroducing the nucleic acid molecule of claim 12 into a non-cell systemfor transcription and/or translation of a nucleic acid molecule,incubating the non-cell system under conditions sufficient fortranscription and/or translation of the nucleic acid molecule andisolating the polypeptide produced by the transcription and/ortranslation from the non-cell system.
 18. A method for making a Smad7polypeptide comprising introducing the expression vector of claim 14into a non-cell system for transcription and/or translation of a nucleicacid molecule, incubating the non-cell system under conditionssufficient for transcription and/or translation of the nucleic acidmolecule and isolating the polypeptide produced by the transcriptionand/or translation from the non-cell system.